Patent Application
20140309278
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 11, 2014, is named 112172-200_SL.txt and is 26,368 bytes in size.
This invention relates to cancer therapy, and more specifically, to combination cancer therapy utilizing microRNAs and EGFR-TKI inhibitors.
Lung cancer accounts for the most cancer-related deaths in both men and women. An estimated ˜220,000 new cases of lung cancer are expected in 2012, accounting for about 14% of all cancer diagnoses (Cancer Facts & Figures 2012, Society). Lung cancer is the leading cause of cancer-related deaths totaling in an estimated 160,000 deaths in 2012 which equals about 28% of all cancer deaths. Lung cancers are divided into two major classes. Small cell lung cancer (SCLC) affects 20% of patients and non-small cell lung cancer (NSCLC) affects approximately 80%. NSCLC consists of three major types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, with lung adenocarcinomas and squamous cell carcinomas accounting for the vast majority of all lung cancers (see, e.g., Forgacs et al., Pathol Oncol Res, 2001. 7(1):6-13; Sekido et al., Biochim Biophys Acta, 1998. 1378(1): F21-59). Treatments include surgery, radiation, therapy, chemotherapy, and targeted therapies. For localized NSCLC, surgery is usually the treatment of choice, and survival for most of these patients improves by giving chemotherapy after surgery. Targeted therapies are used depending on the cancer genotype or stage of disease and include bevacizumab (Avastin™, Genentech/Roche), a humanized monoclonal antibody targeting VEGF-A, erlotinib (Tarceva™, Genentech/Roche), an EGFR tyrosine kinase inhibitor (EGFR-TKI), and crizotinib (Xalkori™, Pfizer), an inhibitor of ALK (anaplastic lymphoma kinase) and ROS1 (c-ros oncogene, receptor tyrosine kinase). Crizotinib has been approved by the FDA to treat certain late-stage (locally advanced or metastatic) non-small cell lung cancers and is limited to those that express the mutated ALK gene. Bevacizumab has been first approved for use in first-line advanced non-squamous NSCLC in combination with carboplatin/paclitaxel chemotherapy. Since then, the National Comprehensive Cancer Network recommends bevacizumab as standard first-line treatment in combination with any platinum-based chemotherapy, followed by maintenance bevacizumab until disease progression (Sandler et al., N Engl J Med, 2006. 355(24): 2542-50).
Erlotinib received fast-track approval from the US Food and Drug Administration (FDA) for patients with NSCLC after failure of prior conventional chemotherapy regimen (Cohen et al., Oncologist, 2005. 10(7):461-6; Cohen et al., Oncologist, 2003. 8(4):303-6. It is a reversible inhibitor of the EGFR kinase, designed to act as competitive inhibitors of ATP-binding at the active site of the EGFR kinase (Sharma et al. Nat Rev Cancer, 2007. 7(3):169-81). Gefitinib is another EGFR-TKI agent used in countries outside the US. Although no direct comparative effectiveness trials exist that have compared gefitinib with erlotinib, the data suggest that there are no major therapeutic differences between them (Pao et al., Nat Rev Cancer, 2010. 10(11): 760-74). Early clinical trials using EGFR-TKIs were modestly encouraging with partial responses observed in approximately 10-20% of treated patients with NSCLC (Fukuoka et al. J Clin Oncol, 2003. 21(12):2237-46). A drug response occurred more frequently in females, never-smokers, patients of Asian ethnicity, and those diagnosed with adenocarcinoma or bronchioalveolar histology Fukuoka et al., J Clin Oncol, 2003. 21(12):2237-46; Bell et al., J Clin Oncol, 2005. 23(31):8081-92). Notably, both drugs extend overall patient survival benefit by only ˜2 months, they lose their efficacy due to primary or acquired, secondary resistance (Sharma, supra; Shepherd et al., N Engl J Med, 2005. 353(2):123-32).
The dissatisfactory response rate of gefitinib and erlotinib has triggered multiple studies to assess the genetic background of responsive vs. resistant patient populations. Retroactive analyses of clinical trials revealed that EGFR expression levels did not correlate with a response to gefitinib (Bell, supra). Instead, patients responding to the drugs frequently harbored activating mutations in the EGFR kinase domain (id.). However, less than 50% of patients with EGFR mutations developed a response, indicating the presence of additional factors that determine susceptibility to EGFR-TKIs. Primary resistance or secondary resistance has been associated with (1) K-RAS mutations that may co-exist with EGFR mutations despite the fact that K-RAS and EGFR mutations appeared to be predominantly mutually exclusive (Gazdar et al., Trends Mol Med, 2004. 10(10):481-6; Pao et al., PLoS Med, 2005. 2(1):e17); (2) amplification and overexpression of c-Met, a receptor tyrosine kinase that signals into the PI3K pathway, substituting for an inactivation of EGFR (Engelman et al., Science, 2007. 316(5827):039-43); (3) the acquisition of a second mutation in the catalytic domain of EGFR (usually T790M) (Pao et al. PLoS Med, 2005. 2(3):e73., (4) BRAF mutations (Pratilas et al., Cancer Res, 2008. 68(22):9375-83); (5) ALK translocations (Shaw et al., J Clin Oncol, 2009. 27(26):4247-53); (6) hepatocyte growth factor (HGF) overexpression, the ligand of the MET receptor (Yano et al., Cancer Res, 2008. 68(22):9479-87); (7) the presence of other EGFR mutations (small insertions or duplications in exon 20: D770_N771, ins NPG, ins SVQ, ins G and N771T) (Wu et al., Clin Cancer Res, 2008. 14(15): 4877-82); and (8) genetic lesions that affect signaling downstream of EGFR, including PIK3CA (Engelman et al., J Clin Invest, 2006. 116(10):2695-706; Kawano et al., Lung Cancer, 2006. 54(2):209-15), loss of PTEN (Sos et al., Cancer Res, 2009. 69(8):3256-61), IGF1R and KDM5A (Gong et al., PLoS One, 2009. 4(10) e7273; Sharma et al., Cell. 141(1):69-80). The T790M mutation is found in ˜50% of EGFR-mutant tumors with acquired resistance; KRAS mutations occur in 15-25% of all NSCLCs; and mutated BRAF and ALK translocations are found in 2-3% and 5% of NSCLCs, respectively (Pao et al., Nat Rev Cancer, 2010. 10(11):760-74). Hence, the percentage on NSCLC patients that is likely to respond to EGFR-TKI therapy is relatively small. Additional yet unidentified molecular determinants may exist, which mediate resistance to EGFR inhibitors.
The modest efficacy of erlotinib as single therapeutic agents calls for the combinatorial use of these EGFR-TKIs with other therapeutic regimes. The Phase III clinical trials TRIBUTE/TALENT trials, investigating the effect of erlotinib in combination with cisplatin/gemcitabine or carboplatin/paclitaxel, failed to demonstrate a survival benefit of the drug over the conventional chemotherapies alone (Sharma, supra; Herbst et al., J Clin Oncol, 2005. 23(25):5892-9 and Giaccone et al., J Clin Oncol, 2004. 22(5):777-84 and Herbst et al., J Clin Oncol, 2004. 22(5):785-94. Therefore, erlotinib is currently being tested in combination with other targeted small molecule inhibitors that show promising results in preclinical studies, such inhibitors against mTOR and MET (Pao, supra). Whether this strategy is efficacious in patients with EGFR-TKI resistance remains to be established. Available data suggest that resistant tumors arise from rare cells in untreated tumors already harboring mutations in resistance genes, and that these subpopulations are selected for over the course of TKI treatment (id.). It is also possible that already untreated tumors display a heterogenic profile of EGFR-TKI resistant cells, suggesting that a single drug combination of targeted therapies will not be sufficient for effective treatment. Instead, the sequential use of several combinations might be necessary to eliminate resistant tumors that undergo a positive selection during the prior treatment.
Therefore, despite advances in the treatment of lung cancer, the survival rate of lung cancer patients remains extremely poor. Current targeted therapies, such as EGFR-TKIs, hold considerable promise but lack satisfactory efficacy in monotherapy due to the existence or development of primary and secondary resistance. The combined use of EGFR inhibitors with other targeted treatments may aid in the efficacy of EGFR inhibitors and may help overcome or prevent drug resistance.
Preliminary studies indicate that certain miRNAs can sensitize cancer cells in vitro (reviewed in Bommer et al., Curr Biol, 2007. 17(15):1298-307). For instance, let-7 is able to sensitize lung cancer cells to TRAIL-based, gemcitabine or radiation therapies (Li et al., Cancer Res, 2009. 69(16): 6704-12; Ovcharenko et al., Cancer Res, 2007. 67(22): 10782-8; Weidhaas et al., Cancer Res, 2007. 67(23):11111-6). Similarly, miR-34 enhances the efficiency of conventional therapies in cancer cell lines of the prostate, colon, brain, stomach, bladder and pancreas (Fujita et al., Biochem Biophys Res Commun, 2008. 377(1):114-9; Ji et al., PLoS One, 2009. 4(8):e6816; Kojima et al., Prostate. 70(14):1501-12. Akao et al., Cancer Lett. 300(2):197-204; Weeraratne et al., Neuro Oncol. 13(2):165-75; Ji et al., BMC Cancer, 2008. 8:266; and Vinall et al., Int J Cancer, 2011. 130(11): 2526-38). However, a demonstration for any erlotinib/miRNA combination in cell and animal models of lung cancer remains absent.
Recently, Zong et al. (Chemico-Bio Interac. 2010, 184:431-438) have tested let-7a, miR-126 and miR-145 for their ability to sensitize Gefitinib-resistant cells lines A549 and H460 to gefitinib. The biggest reduction of IC50 was achieved by miR-126 in H440 cells (˜7-fold), whereas the remaining conditions resulted in only 2-3-fold IC50 reductions (see Table 2 in Zhong, supra).
The invention is based, in part, on the discovery that certain microRNAs can be consistently up- or down-regulated in EGFR-TKI-resistant cell lines, and that specific combinations of microRNAs and EGFR-TKI agents can have advantageous and/or unexpected results, for example because they are particularly efficacious in treating certain cancer cells (e.g., synergize, or have greater that additive effect). Accordingly, the invention, in various aspects and embodiments includes contacting cells, tissue, and/or organisms with specific combinations of microRNAs and EGFR-TKI agents. More particularly, the invention can include contacting cancer cells, cancer tissue, and/or organisms having cancer with such combinations of microRNAs and EGFR-TKI agents. The methods can be experimental, diagnostic, and/or therapeutic. The methods can be used to inhibit, or reduce the proliferation of, cells, including cells in a tissue or an organism. The microRNAs can be, for example, mimics or inhibitors of microRNAs that are consistently down- or up-regulated in EGFR-TKI-resistant cells lines.
Accordingly, in various aspects and embodiments, the invention provides methods of treating a subject having a cancer. In certain embodiments, the methods comprise: administering an EGFR-TKI agent to the subject, and administering a microRNA mimic of miR-34, miR-126, miR-124, miR-147, and miR-215 to the subject. Similar methods include contacting (e.g., treating) a cell or tissue (e.g., a cancer cell or cancer tissue such as a tumor) with an EGFR-TKI agent, and contacting the cell or tissue with a microRNA mimic of miR-34, miR-126, miR-124, miR-147, and miR-215. The microRNA can comprise a sequence that is at least 80% (or 85, 90, 95, 100%) identical to at least one of SEQ ID NOs:1-6 and 168-179 (miR-34, miR-126, miR-124, miR-147, and miR-215, as well as family members, functional homologs, seed sequences, or consensus sequences thereof). These, and other, microRNAs can comprise natural nucleic acids, derivatives and chemically modified forms thereof, as well as nucleic acid analogs.
In various aspects and embodiments, the invention provides methods of administering an EGFR-TKI agent to a subject (e.g., a subject having cancer), and administering a microRNA mimic of a microRNAs listed in Appendix A as SEQ ID NOs:8-122 (downregulated microRNAs) to the subject. Similar methods include contacting a cell or tissue (e.g., a cancer cell or cancer tissue such as a tumor) with an EGFR-TKI agent, and contacting the cell or tissue with a microRNA mimic of a microRNAs listed in Appendix A as SEQ ID NOs:8-122 (downregulated microRNAs). The microRNA can comprise a sequence that is at least 80% (or 85, 90, 95, 100%) identical to at least one of SEQ ID NOs:8-122.
In various aspects and embodiments, the invention provides methods of administering an EGFR-TKI agent to a subject (e.g., a subject having cancer), and administering an inhibitor of a microRNAs listed in Appendix A as SEQ ID NOs:123-167, preferably, SEQ ID NOs:156-167, more preferably, SEQ ID NOs:159, 164, and 165 (upregulated microRNAs). Similar methods include contacting a cell or tissue (e.g., a cancer cell or cancer tissue such as a tumor) with an EGFR-TKI agent, and contacting the cell or tissue with an inhibitor of a microRNAs listed in Appendix A as SEQ ID NOs:123-167, preferably, SEQ ID NOs:156-167, more preferably, SEQ ID NOs:159, 164, and 165 (upregulated microRNAs). The inhibitor can be a microRNA comprising a sequence that is at least 80% (or 85, 90, 95, 100%) complementary to the microRNA.
In various embodiments, the EGFR-TKI agent can be erlotinib or an analogous EGFR-TKI agent such as gefitinib, afatinib, panitumumab, or cetuximab, or a HER2 inhibitor such as lapatinib, pertuzumab, or trastuzumab. In some embodiments, the EGFR inhibitor is erlotinib and the microRNA is at least 80% (or 85, 90, 95, 100%) identical to one of SEQ ID NOs:1-4, for example SEQ ID NO:1.
In various embodiments, the cancer can be a cancer in which combinations of microRNAs and EGFR-TKI inhibitors in accordance with the present invention are effective therapeutics, for example lung cancer (e.g., non-small cell lung, NSCL) and liver cancer (e.g., hepatocellular carcinoma, HCC). The cancer can include a metastatic lesion in the liver.
In various embodiments, the cancer can be is resistant to treatment with the EGFR-TKI agent alone. The resistance can be primary or secondary (acquired). The cancer can be a lung (e.g., NSCL) cancer that has primary or secondary resistance to treatment with the EGFR-TKI agent alone. The cancer can be a liver cancer (e.g., HCC) that has primary or secondary resistance to treatment with the EGFR-TKI agent alone.
In various embodiments, the EGFR-TKI agent can be administered at an effective dose that is below (e.g., at least 50% below) the dose needed to be effective in the absence of the microRNA administration. The dose can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90% before the dose necessary in absence of the microRNA.
In various embodiments, the IC50 of the EGFR-TKI agent is reduced (e.g., at least 2-fold) relative to the IC50 in the absence of the microRNA administration. The IC50 can be reduced by at least 1.5, 2, 2.5, 3, 4, 5, or 10 fold.
In various embodiments, the subject is a human, non-human primate, or laboratory animal (e.g., mouse, rat, guinea pig, rabbit, pig). The subject can have a KRAS mutation. The subject can have a EGFR mutation. In some embodiments, the subject has a primary or secondary resistance to erlotinib, for example, a patient who has developed or is likely to develop resistance to an EGFR-TKI agent. Alternatively, the subject's cancer may be sufficiently sensitive to the EGFR-TKI agent, however, that toxicity of the monotherapy may indicate that a lower dose of EGFR-TKI agent is desirable.
Various aspects, embodiments, and features of the invention are presented and described in further detail below. However, the foregoing and following descriptions are illustrative and explanatory only and are not restrictive of the invention, as claimed.
FIGS. 8 and 8A-C illustrates endogenous miR-34 and mRNA levels of genes controlling erlotinib resistance in NSCLC cells.
FIGS. 9 and 9A-B illustrates dose-response curves of the single agents in NSCLC cells resistant to erlotinib.
FIGS. 10 and 10A-D illustrates summary tables showing potency, CI and DRI values of erlotinib and miR-34a combined at various concentrations and ratios in NSCLC cells. Combinations that yield Fa>65%, CI<0.6, DRI>2 are highlighted in grey and are considered relevant.
FIGS. 12 and 12A-B illustrates dose-response curves of the single agents in HCC cells resistant to erlotinib.
FIGS. 13 and 13A-D illustrates summary tables showing potency, CI and DRI values of erlotinib and miR-34a combined at various concentrations and ratios in HCC cells.
The invention is based, in part, on the discovery that certain microRNAs can be consistently up- or down-regulated in EGFR-TKI-resistant cell lines, and that specific combinations of microRNAs and EGFR-TKI agents can have advantageous and/or unexpected results, for example because they are particularly efficacious in treating certain cells (e.g., synergize, or have greater that additive effect). Accordingly, the invention, in various aspects and embodiments includes contacting cells, tissue, and/or organisms with specific combinations of microRNAs and EGFR-TKI agents. More particularly, the invention can include contacting cancer cells, cancer tissue, and/or organisms having cancer with such combinations of microRNAs and EGFR-TKI agents. The methods can be experimental, diagnostic, and/or therapeutic. The methods can be used to inhibit, or reduce the proliferation of, cells, including cells in a tissue or an organism. The microRNAs can be, for example, mimics or inhibitors of microRNAs that are consistently down- or up-regulated in EGFR-TKI-resistant cells lines.
microRNAs
microRNAs (miRNAs) are small non-coding, naturally occurring RNA molecules that post-transcriptionally modulate gene expression and determine cell fate by regulating multiple gene products and cellular pathways (Bartel, Cell, 2004. 116(2):281-97) miRNAs interfere with gene expression by either degrading the mRNA transcript by blocking the protein translation machinery (Bartel, supra) miRNAs target mRNAs with sequences that are fully or merely partially complementary which endows these regulatory RNAs with the ability to target a broad but nevertheless specific set of mRNAs. To date, there are 1,500 human annotated miRNA genes with roles in processes as diverse as cell proliferation, differentiation, apoptosis, stem cell development, and immune function (Costinean et al., Proc Natl Acad Sci USA, 2006. 103(18):7024-9). Often, the misregulation of miRNAs can contribute to the development of human disease including cancer (Esquela-Kerscher et al., Nat Rev Cancer, 2006. 6(4):259-69; Calin et al., 2006. 6(11):857-66) miRNAs deregulated in cancer can function as bona fide tumor suppressors or oncogenes. A single miRNA can target multiple oncogenes and oncogenic signaling pathways (Forgacs et al., Pathol Oncol Res, 2001. 7(1):6-13), and translating this ability into a future therapeutic may hold the promise of creating a remedy that is effective against tumor heterogeneity. Thus, miRNAs have the potential of becoming powerful therapeutic agents for cancer (Volinia et al., Proc Natl Acad Sci USA, 2006. 103(7):2257-61; Tong et al., Cancer Gene Ther, 2008. 15(6):341-55) that act in accordance with our current understanding of cancer as a “pathway disease” that can only be successfully treated when intervening with multiple cancer pathways (Wiggins et al., Cancer Res, 2010. 70(14): 5923-5930.; Jones et al., Science, 2008. 321(5897):1801-6; Parsons et al., Science, 2008. 321(5897):1807-12).
As of March 2013, Mirna Therapeutics (Austin, Tex.) has completed the preclinical development program to support the manufacture of cGMP-materials and the conduction of IND-enabling studies for a miR-34-based supplementation therapy (MRX34). Mirna evaluated the toxicity as well as the pharmacokinetic profile of the formulation containing miR-34 mimic in non-GLP pilot studies using mice, rats and non-human primates. These experiments did not show adverse events at the predicted therapeutic levels of MRX34, as measured by clinical observations, body weights, clinical chemistries (including LFT, RFT and others), hematology, gross pathology, histopathology of select organs and complement (CH50). In addition, miRNA mimics formulated in lipid nanoparticles do not induce the innate immune system as demonstrated in fully immunocompetent mice, rats, non-human primates, as well as human whole blood specimens. A more detailed review of the pre-clinical data is provided in Bader, Front Genet. 2012; 3:120.
In methods of the inventions, a specific microRNA (e.g., synthetic microRNA mimic or inhibitor) is administered to a subject as part of a combination therapy with an EGFR-TKI agent. In specific embodiments, such a microRNA is selected from the group consisting of SEQ ID NOs:1-179. These microRNAs are well known in the art, and one of skill in the art would understand that they include the conventional naturally occurring sequences (provided herein) and any chemically modified versions and sequence homologues thereof.
In various aspects and embodiments, the present invention employs a microRNA mimic or inhibitor, which is not delivered through transfection into a cell. Rather, in various embodiments, the microRNA can be administered by methods such as injection or transfusion. In some embodiments, rather than an isolated cell, tissue, or culture thereof, the subject can be a mammal (e.g., a human or laboratory animal such as a mouse, rat, guinea pig, rabbit, pig, non-human primate, and the like).
The microRNAs used in connection with the invention can be 7-130 nucleotides long, double stranded RNA molecules, either having two separate strands or a hairpin structure. For example, a microRNA can be 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 7-30, 7-25, 15-30, 15-25, 17-30, or 17-25 nucleotides long. One of the two strands, which is referred to as the “guide strand”, contains a sequence which is identical or substantially identical to the seed sequence (nucleotide positions 2-9) of the parent microRNA sequence shown in the table below. “Substantially identical”, as used herein, means that at most 1 or 2 substitutions and/or deletions are allowed. In some embodiments, the guide strand comprises a sequence which is at least 80%, 85%, 90%, 95% identical to the respective full length sequence provided herein. The second of the two strands, which is referred to as a “passenger strand”, contains a sequence that is complementary or substantially complementary to the seed sequence of the corresponding given microRNA. “Substantially complementary”, as used herein, means that at most 1 or 2 mismatches and/or deletions are allowed. In some embodiments, the passenger strand comprises a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to the complement of the respective full length sequence provided herein. In some embodiments, the microRNA is a mimic of miR-34a, miR-34b, miR-34c, miR-449a, miR-449b, miR-449c, miR-192, miR-215, miR-126, miR-124, miR-147, or an analog or homolog thereof. In some embodiments, the microRNA includes the seed sequence of one of these microRNAs.
GGCAGUG
UAAGGCA
CGUACCG
UGUGUGG
UGACCUA
The microRNAs (e.g., microRNA mimics) can be formulated in liposomes such as, for example, those described in U.S. Pat. Nos. 7,858,117 and 7,371,404; US Patent Application Publication Nos. 2009-0306194 and 2011-0009641. Other delivery technologies are known in the art and available, including expression vectors, lipid or various ligand conjugates.
In certain embodiments, methods of the invention include administering an inhibitor of a microRNA selected from the microRNAs listed in Appendix A as SEQ ID NOs:123-167, preferably, SEQ ID NOs:156-167, more preferably, SEQ ID NOs:159, 164, and 165 Inhibitors of microRNA are well known in the art and are typically antisense molecules that are complementary to the target microRNA, however, other types of inhibitors can also be used. Inhibitors of microRNAs are described, for example, in U.S. Pat. No. 8,110,558. In certain embodiments, an inhibitor of a microRNA contains a 9-20, 10-18, or 12-17 nucleotide long sequence that is complementary or substantially complementary to the corresponding upregulated microRNA sequence listed in Appendix A as SEQ ID NOs:123-167, preferably, SEQ ID NOs:156-167, more preferably, SEQ ID NOs:159, 164, and 165.
microRNAs and their inhibitors can also be chemically modified, for example, microRNAs may have a 5′ cap on the passenger strand (e.g., NH2—(CH2)6—O—) and/or a mismatch at the first and/second nucleotide of the same strand. Other possible chemical modifications can include backbone modifications (e.g., phosphorothioate, morpholinos), ribose modifications (e.g., 2′-OMe, 2′-Me, 2′-F, 2′-4′-locked/bridged sugars (e.g., LNA, ENA, UNA) as well as nucleobase modifications (see, e.g., Peacock et al, 2011. J Am Chem Soc., 133(24):9200-9203. In certain embodiments, the microRNAs, and in particular, miR-34 and miR-124 have modifications as described in U.S. Pat. No. 7,960,359 and US Patent Application Publication Nos. 2012-0276627 and 2012-0288933.
microRNAs can be administered intravenously as a slow-bolus injection at doses ranging 0.001-10.0 mg/kg per dose, for example, 0.01-3.0, 0.025-1.0 or 0.25-0.5 mg/kg per dose, with one, two, three or more doses per week for 2, 4, 6, 8 weeks or longer as necessary.
Methods of the invention involve administering an EGFR-TKI agent to a subject. The family of epidermal growth factor receptors (EGFR) comprises four structurally related cell-surface receptor tyrosine kinases that bind and elicit functions in response to members of the epidermal growth factor (EGF) family. In humans, this includes EGFR, also known as Her-1 and ErbB1, Her-2, also referred to as Neu and ErbB2, Her-3 (ErbB3), and Her-4 (ErbB4). Hyperactivation of ErbB signaling is associated with the development of a wide variety of solid tumors. Accordingly, in various additional embodiments, the present invention includes combinations of microRNAs with erlotinib as well as other EGFR inhibitors, such as gefitinib, afatinib, panitumumab and cetuximab, as well as HER2 inhibitors such as lapatinib, pertuzumab and trastuzumab.
In certain embodiments, the EGFR-TKI is erlotinib, the active ingredient of the drug currently marketed under the trade name TARCEVA®. Unless expressly stated otherwise, the term “erlotinib” herein refers the compound of Formula I, as well as to any of its salts or esters thereof.
Erlotinib is a tyrosine kinase inhibitor, a quinazolinamine with the chemical name N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine. In specific embodiments, the erlotinib is erlotinib hydrochloride. TARCEVA® tablets for oral administration are available in three dosage strengths containing erlotinib hydrochloride (27.3 mg, 109.3 mg and 163.9 mg) equivalent to 25 mg, 100 mg and 150 mg erlotinib and the following inactive ingredients: lactose monohydrate, hypromellose, hydroxypropyl cellulose, magnesium stearate, microcrystalline cellulose, sodium starch glycolate, sodium lauryl sulfate and titanium dioxide. The tablets also contain trace amounts of color additives, including FD&C Yellow #6 (25 mg only) for product identification. Further information is available from the approved drug label.
Erlotinib is also described in U.S. Pat. No. 6,900,221, herein incorporated by reference, and the corresponding PCT Publication WO 01/34574.
The approved recommended dose of TARCEVA® for NSCLC is 150 mg/day; the approved dose for pancreatic cancer is 100 mg/day. Doses may be reduced in 50 mg decrements when necessary.
In certain embodiments where the EGFR-TKI agent is erlotinib, the microRNA does not have the sequence of miR-126 (e.g., less that 100, 95, 90, 85, or 80% identity with the sequence of human miR-126 or seed sequence thereof).
In other embodiments, the EGFR-TKI agent is gefitinib, the active ingredient of the drug marketed under the trade name IRESSA®. Unless expressly stated otherwise, the term “gefitinib” refers herein the compound of Formula II, as well as to any of salts or esters thereof.
Gefitinib is a tyrosine kinase inhibitor with the chemical name 4-quinazolinamine, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4-morpholin) propoxy], and also is known as ZD1839. The clinical formulation is supplied as 250 mg tablets, containing the active ingredient, lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, povidone, sodium lauryl sulfate and magnesium stearate. The recommended dose as a single therapy is one 250 mg tablet per day. Further information can be found on the approved drug label.
Other EGFR inhibitors, such as afatinib, panitumumab and cetuximab, as well as HER2 inhibitors such as lapatinib, pertuzumab and trastuzumab are known in the art and, thus, a person of ordinary skill would readily know their structure, formulation, dosing, and administration, etc. (e.g., based on published medical information such as an approved drug label) as would be required in use with the present invention.
The invention provides methods and compositions for treating cancer cells and/or tissue, including cancer cells and/or tissue in a subject, or in vitro treatment of isolated cancer cells and/or tissue. If in a subject, the subject to be treated can be an animal, e.g., a human or laboratory animal.
The subject being treated may have been diagnosed with cancer, for example, lung cancer (non-small cell lung cancer (NSCLC), e.g., adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), pancreatic cancer, or cancer in the liver, or any other type of cancer that benefits from a EGRF inhibition, including breast cancer, HCC, colorectal cancer, head and neck cancers, prostate, brain, stomach, or bladder cancer. In some embodiments, the cells or the subject have/has a primary or secondary resistance to an EGFR-TKI agent.
The subject may have locally advanced, unresectable, or metastatic cancer and/or may have failed a prior first-line therapy. In some embodiments, the subject has undergone a prior treatment with an EGRR-TKI agent lasting at least 2, 4, 6, 8, 10 months or longer. In other embodiments, the subject has the T790M mutation in EGFR (Balak et al. 2006. Clin Cancer Res, 12(1):6494-501). In other embodiments, the subjects are patients who have experienced one or more significant adverse side effect to an EGFR-TKI agent and therefore require a reduction in dose. The subject being treated may also be the one characterized by one of the following: (1) K-RAS mutation; (2) amplification and overexpression of c-Met; (3) BRAF mutation; (4) ALK translocation (5) hepatocyte growth factor (HGF) overexpression; (6) other EGFR mutations (small insertions or duplications in exon 20: D770_N771, ins NPG, ins SVQ, ins G and N771T; and (7) genetic lesions that affect signaling downstream of EGFR, including PIK3CA, loss of PTEN, IGF1R and KDM5A.
In various embodiments, the cancer is liver cancer (e.g., HCC). The liver cancer may not be resistant to an EGFR-TKI agent. Alternatively, the liver cancer (e.g., HCC) can have primary or secondary resistance to an EGFR-TKI agent. The subject can be a responder to an EGFR-TKI agent in the absence of the microRNA. The subject can be a non-responder to a EGFR-TKI in the absence of the microRNA. In some embodiments, the subject has undergone a prior treatment with the EGFR-TKI agent lasting at least 2, 4, 6, 8, 10 months or longer. In other embodiments, the subjects are patients who have experienced one or more significant adverse side effect to the EGFR-TKI agent and therefore require a reduction in dose.
In various embodiments, the liver cancer (e.g., HCC) can be intermediate, advanced, or terminal stage. The liver cancer (e.g., HCC) can be metastatic or non-metastatic. The liver cancer (e.g., HCC) can be resectable or unresectable. The liver cancer (e.g., HCC) can comprise a single tumor, multiple tumors, or a poorly defined tumor with an infiltrative growth pattern (into portal veins or hepatic veins). The liver cancer (e.g., HCC) can comprise a fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell), or clear cell pattern. The liver cancer (e.g., HCC) can comprise a well differentiated form, and tumor cells resemble hepatocytes, form trabeculae, cords, and nests, and/or contain bile pigment in cytoplasm. The liver cancer (e.g., HCC) can comprise a poorly differentiated form, and malignant epithelial cells are discohesive, pleomorphic, anaplastic, and/or giant. In some embodiments, the liver cancer (e.g., HCC) is associated with hepatits B, hepatitis C, cirhhosis, or type 2 diabetes.
In some embodiments, the therapeutically effective dose of an EGFR-TKI agent is reduced. For example, the weekly or monthly dose of the EGFR-TKI agent reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to the maximum recommended dose or the maximum tolerated dose. In other embodiments, the EGFR-TKI agent is administered at an effective dose that at least 50%, 60%, 70%, 80%, 90% or more below the dose needed to be effective in the absence of the microRNA (or microRNA inhibitor) administration. For example, erlotinib can be administered at a dose of 50, 40, 30, 25 mg per day or less. In some embodiments, the IC50 of an EGFR-TKI agent is reduced by at least 4-, 5-, 10-, 20-, 30-, 40-, 50-, or 100-fold relative to the IC50 in the absence of the microRNA treatment (or microRNA inhibitor treatment if the inhibitor is to be administered). IC50 can be determined, for example, as illustrated in the Examples.
Combination chemotherapy or polytherapy is the use of more than one medication or other therapy (e.g., as opposed to monotherapy, which is any therapy taken alone). As used herein with reference to the present invention, the term refers to using specific combinations of EGFR-TKI agents and microRNAs.
As used herein for describing ranges, e.g., of ratios, doses, times, and the like, the terms “about” embraces variations that are within the relevant margin of error, essentially the same (e.g., within an art-accepted confidence interval such as 95% for phenomena that follow a normal or Gaussian distribution), or otherwise does not materially change the effect of the thing being quantified.
The EGFR-TKI agent dosing amount and/or schedule can follow clinically approved, or experimental, guidelines. Further to the description in the EGFR-TKI agents section, in various embodiments, the dose of EGFR-TKI agent can be a dose prescribed by the FDA drug label, or label/instructions of another agency.
Likewise the microRNA dosing amount and/or schedule can follow clinically approved, or experimental, guidelines. In various embodiments, the dose of microRNA is about 10, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, or 250 mg/m2 per day.
In various embodiments the microRNA is administered to the subject in 1, 2, 3, 4, 5, 6, or 7 daily doses over a single week (7 days). The microRNA can be administered to the subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 daily doses over 14 days. The microRNA can be administered to the subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 daily doses over 21 days. The microRNA can be administered to the subject in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 daily doses over 28 days.
In various embodiments the microRNA is administered for: 2 weeks (total 14 days); 1 week with 1 week off (total 14 days); 3 consecutive weeks (total 21 days); 2 weeks with 1 week off (total 21 days); 1 week with 2 weeks off (total 21 days); 4 consecutive weeks (total 28 days); 3 consecutive weeks with 1 week off (total 28 days); 2 weeks with 2 weeks off (total 28 days); 1 week with 3 consecutive weeks off (total 28 days).
In various embodiments the microRNA is: administered on day 1 of a 7, 14, 21 or 28 day cycle; administered on days 1 and 15 of a 21 or 28 day cycle; administered on days 1, 8, and 15 of a 21 or 28 day cycle; or administered on days 1, 2, 8, and 15 of a 21 or 28 day cycle. The microRNA can be administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.
A course of EGFR-TKI agent-microRNA therapy can be prescribed by a clinician. The combination therapy can be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles.
A course of EGFR-TKI agent-microRNA therapy can be continued until a clinical endpoint is met. In some embodiments, the therapy is continued until disease progression or unacceptable toxicity occurs. In some embodiments, the therapy is continued until achieving a pathological complete response (pCR) rate defined as the absence of cancer. In some embodiments, the therapy is continued until partial or complete remission of the cancer. Administering the microRNA and the EGFR-TKI agent to a plurality of subject having cancer may increase the Overall Survival (OS), the Progression free Survival (PFS), the Disease Free Survival (DFS), the Response Rate (RR), the Quality of Life (QoL), or a combination thereof.
In various embodiments, the treatment reduces the size and/or number of the cancer tumor(s); prevent the cancer tumor(s) from increasing in size and/or number; and/or prevent the cancer tumor(s) from metastasizing.
In the methods of the invention, administration is not necessarily limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration to an individual may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques, dosages, and excipients are well known to persons skilled in the art (see, e.g., Physicians' Desk Reference (PDR®) 2005, 59th ed., Medical Economics Company, 2004; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al. 21th ed., Lippincott, Williams & Wilkins, 2005).
Additionally, effective dosages achieved in one animal may be extrapolated for use in another animal, including humans, using conversion factors known in the art. See, e.g., Freireich et al., Cancer Chemother Reports 50(4):219-244 (1966) and Table 2 for equivalent surface area dosage factors). Reports 50(4):219-244 (1966) and Table 2 for equivalent surface area dosage factors).
In various embodiments, the microRNA is administered prior to the EGFR-TKI agent, concurrently with the EGFR-TKI agent, after the EGFR-TKI agent, or a combination thereof. The microRNA can be administered intravenously. The microRNA can be administered systemically or regionally.
The combination therapies of the invention are not specifically limited to any particular course or regimen and may be employed separately or in conjunction with other therapeutic modalities (e.g., chemotherapy or radiotherapy).
A combination therapy in accordance with the present invention can include additional therapies (e.g., pharmaceutical, radiation, and the like) beyond the EGFR-TKI agent and microRNA. Similarly, the present invention can be used as an adjuvant therapy (e.g., when combined with surgery). In various embodiments, the subject is also treated by surgical resection, percutaneous ethanol or acetic acid injection, transcatheter arterial chemoembolization, radiofrequency ablation, laser ablation, cryoablation, focused external beam radiation stereotactic radiotherapy, selective internal radiation therapy, intra-arterial iodine-131-lipiodol administration, and/or high intensity focused ultrasound.
The combination of the microRNA and EGFR-TKI agent can be used as an adjuvant, neoadjuvant, concomitant, concurrent, or palliative therapy. The combination of the microRNA and EGFR-TKI agent can be used as a first line therapy, second line therapy, or crossover therapy.
In some embodiments, the therapeutically effective dose of EGFR-TKI agent is reduced through combination with the microRNA. For example, the daily, weekly, or monthly dose of EGFR-TKI agent can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to the maximum recommended dose or the maximum tolerated dose. In other embodiments, EGFR-TKI agent is administered at an effective dose that at least 50%, 60%, 70%, 80%, 90% or more below the dose needed to be effective in the absence of the microRNA (or microRNA inhibitor) administration. In some embodiments, the IC50 of EGFR-TKI agent is reduced by at least 4-, 5-, 10-, 20-, 30-, 40-, 50-, or 100-fold relative to the IC50 in the absence of the microRNA (or microRNA inhibitor).
Further description and embodiments of combination therapies are provided in the Examples section below.
As discussed and further illustrated in the examples below, the present invention provides methods and compositions for treating cancer (e.g., lung or liver cancer) where the EGFR-TKI agent and microRNA are administered in a combination that is particularly effective (e.g., synergistic or more than additive). While synergy and synonymous terms are commonly used in the art, the property is not always defined or quantified (and, hence, the purported synergy may not actually be present). In connection with the present invention and the examples below, combination index (CI) values were used to quantify the effects of various combinations of EGFR-TKI agent and microRNA.
In various embodiments, the combination of EGFR-TKI agent and microRNA exhibits a CI<1 in the cancer (e.g., lung cancer or liver cancer). The combination can exhibits a CI<0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, or 0.20 in the cancer).
The following examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The Examples do not in any way limit the invention.
We followed a protocol described in Engelman et al. (supra) to generate NSCLC lines with acquired resistance to erlotinib. Briefly, parental HCC827 cells highly sensitive to erlotinib (IC50erlo=0.054 μM) were incubated with erlotinib at increasing concentrations over 10 weeks until cells were able to proliferate in medium containing erlotinib at a concentration that is equivalent to IC90 in parental HCC827 cells. Over the course of the selection, 3 cell lines from individual cell clones were obtained (HCC827clone 5,6,7). In addition, we obtained a heterogenic mass culture presumably originating from multiple clones (HCC827res.pool) (see
Table 3 provides the list of 4 NSCLC cells used to assess the combinatorial effects of miRNAs and EGFR-TKIs. The particular cell lines were selected based on the IC50 values of EGFR-TKIs in these cells, their oncogenic properties and their susceptibility to miRNAs. This list includes cell lines that are resistant to erlotinib, and cells that are sensitive. The IC50 values of erlotinib for each of these cell lines as reported in the scientific literature are shown. In these examples, cell lines with IC50 values>1 μM are considered resistant.
All four cell lines, as well as the parental HCC827 line were used for RNA extraction and subjected to mRNA (Affymetrix HG-U133 Plus 2.0) and miRNA (Agilent/Sanger12—0) array analysis. Unexpectedly, relatively few mRNAs were differentially expressed between resistant and parental lines (data not shown). In contrast, expression levels of miRNAs were significantly altered. A comparison of miRNA expression between the resistant cells and the parental line showed that clone #7 is most closely related to HCC827 (R2=0.9347), and the resistant pool is the least related line (R2=0.8308). This is in accord with the hypothesis that the pool arose from multiple clones. Unsupervised clustering of miRNAs identified 15 upregulated and 23 down-regulated miRNAs across all resistant HCC827 cells when compared to the parental line (
Lung carcinoma cell lines used in the combination studies included cell lines resistant (H1299, H460, HCC827, all resistant) or sensitive (HCC827 parental) to erlotinib. The main aim of the combination was to achieve an enhanced therapeutic effect of erlotinib (decreased IC50) and to reduce the dose and toxicity of erlotinib. The evaluation of the combinatorial work was performed following the “Fixed Concentration Model” (Fiebig, H. H., Combination Studies). The cytotoxic compound A (erlotinib) is tested at 7-8 concentrations, and compound B (miRNA) at one weak concentration. Drug or miRNA effects on cellular proliferation were assessed using AlamarBlue assay (Invitrogen, Carlsbad, Calif.). IC50 values of erlotinib alone and in the combinations were calculated using the GraphPad software.
First, IC50 values of erlotinib alone or miRNAs alone were determined in the cells. miRNAs were reverse transfected at fixed, weak concentration (˜IC25). MicroRNA sequences used were as shown in Table 1. A scrambled sequence was used a negative control. Then, the cells were treated with erlotinib in a serial dilution. Cell proliferation inhibition was analyzed 3 days post drug treatment by AlarmaBlue assay. IC50 values of erlotinib combined with miRNA was determined using the GraphPad software. The combinatorial effect was evaluated by the visual inspection of the dose response curve and a shift of the IC50 value. The IC50 results for erlotinib alone or in combination with each of the six tested miRNAs are reported in Table 4 respectively.
To test effects of the erlotinib/microRNA combinations in vivo, a tumor mouse model is used that, for instance, is based on orthotopic xenografts that stably express a luciferase reporter gene. A typical efficacy study includes 8 animals per group. Next to erlotinib/miRNA combinations, other study groups include erlotinib alone, miRNA alone, as well as erlotinib/miR-NC and no-treatment controls. When tumor lesions in the lung become apparent through IVIS imaging, miRNA treatment is started. miRNAs are administered intravenously every other day complexed in the nanoparticles at a moderately effective dose to allow the detection of erlotinib enhancement (1-10 mg/kg). Erlotinib will be given daily by gavage at a dose of /day which has shown to be well tolerated in mice. Treatment durations are 2-4 weeks, or until control mice become moribund whichever comes first. Animals are monitored closely to detect signs of toxicity. Upon sacrifice, lungs and lung tumor tissues are collected and subjected to histopathological analysis (H&E; ki67 and casp3 IHC if justified). RNA are extracted from normal lung, lung tumors, spleen and whole blood to measure concentrations of miRNA mimics by qRT-PCR. In addition, tumor samples are used to test for knock-down of direct/validated miRNA targets (qRT-PCR). The level of metastases in major organs can be assessed, either by H&E and a human-specific IHC stain (STEM121, StemCells, Inc.).
It is expected that the erlotinib/miRNA combinations show better in vivo efficacy than erlotinib alone with a concurrent repression of known miRNA targets in the tumor tissue. It is also expected that animals treated with erlotinib/miRNA combos are less likely to develop metastases and show improved survival.
This example investigates the relationship of miR-34a and erlotinib and the therapeutic activity of the combination in NSCLC cells with primary and acquired erlotinib resistance. The drug combination was also tested in a panel of hepatocellular carcinoma cells (HCC), a cancer type known to be refractory to erlotinib. Using multiple analytical approaches, drug-induced inhibition of cancer cell proliferation was determined to reveal additive, antagonistic or synergistic effects. The data show a strong synergistic interaction between erlotinib and miR-34a mimics in all cancer cells tested. Synergy was observed across a range of dose levels and drug ratios, reducing IC50 dose requirements for erlotinib and miR-34a by up to 46-fold and 13-fold, respectively. Maximal synergy was detected at dosages that provide a high level of cancer cell inhibition beyond the one that is induced by the single agents alone and, thus, is of clinical relevance. The data shows that a majority of NSCLC and other cancers previously not suited for EGFR-TKI therapy prove sensitive to the drug when used in combination with a micro RNA based therapy.
Materials and Methods
Cell lines: Human non-small cell lung cancer (NSCLC) cell lines A549, H460, H1299, H226, HCC827 parental and HCC827res were used to assess the combinatorial effects of micro RNA and EGFR-TKIs. The particular cell lines were selected based on the high IC50 values of EGFR-TKIs in these cells, their oncogenic properties and susceptibility to miRNAs. These cell lines are either resistant (A549, H460, H1299, H226) or sensitive (HCC827). In addition, cell lines with acquired resistance were created by applying increased selective pressure of erlotinib over ten weeks, starting at an equivalent of IC10 and ending at an IC90 equivalent. As cellular proliferation exhibited normal doubling rates under IC90 selection, the resistant cells were plated at a low dilution (HCC827res) or high dilution to create near-pure, resistant clones (HCC827res-#5, 6 and 7). To study effects in hepatocellular carcinoma (HCC) cells, Hep3B, Huh7, C3A and HepG2 were used. Huh7 cells were acquired from the Japanese Collection of Research Bioresources Cell Bank. All other parental cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va.) and cultured according to the supplier's instructions.
RNA isolation and qRT-PCR: Total RNA from cell pellets was isolated using the mirVANA PARIS RNA isolation kit (Ambion, Austin, Tex.) following the manufacturer's instructions. RNA concentration was determined by absorbance measurement (A260) on a Nanodrop ND-1000 (Thermo Scientific, Wilmington, Del.). For the quantification of miRNA and mRNA by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), we used commercially available reagents. The RNA was converted to cDNA using MMLV-RT (Invitrogen, Carlsbad, Calif.) under the following conditions: 4° C. for 15 min; 16° C. for 30 min; 42° C. for 30 min; 85° C. for 5 min Following cDNA synthesis, qPCR was performed on 2 μL of cDNA on the ABI Prism 7900HT SDS (Applied Biosystems, Life Technologies, Foster City, Calif.) using Platinum Taq Polymerase (Invitrogen) under the following cycling conditions: 95° C. for 1 min (initial denature); then 50 cycles of 95° C. for 5 sec, 60° C. for 30 sec. TaqMan Gene Expression Assays and TaqMan MicroRNA Assays were used for expression analysis of mRNA and miRNA in all lung and liver cell lines. For miRNA expression, additions to the manufacturers' reagents include DMSO (final concentration of 6%) and tetramethylammoniumchloride (TMAC; final concentration of 50 mM in both RT and PCR) to improve the slope, linearity and sensitivity of the miRNA assays. Expression levels of both miRNA and mRNA were determined by relative quantitation to the HCC827 parental cell line. The raw Ct values of the miRNA and mRNA targets were normalized to selected housekeeping genes to create delta-Ct values, converted to linear space and then expressed as percentage expression.
miRNA and EGFR-TKI treatment: Erlotinib hydrochloride was purchased from LC Laboratories (Woburn, Mass.). Synthetic miR-34a and miR-NC mimics were manufactured by Life Technologies (Ambion, Austin, Tex.). To determine the IC50 value of each drug alone, 2,000-3,000 cells per well were seeded in a 96-well plate format and treated with either erlotinib or miR-34a as follows. (i) miR-34a mimics were reverse-transfected in triplicates in a serial dilution (0.03-30 nM) using RNAiMax lipofectamine from Invitrogen. As controls, cells were also transfected with RNAiMax alone (mock) or in complex with a negative control miRNA mimic (miR-NC). Cells were incubated with AlamarBlue (Invitrogen) 4 days or 6 days post transfection to determine cellular proliferation of lung or liver cancer cells, respectively. Proliferation data were normalized to mock-transfected cells. (ii) Erlotinib, prepared as a 10 and 20 mM stock solution in dimethyl sulfoxide (DMSO), was added to cells one day after seeding at a final concentration ranging from 0.1 and 100 μM. Solvent alone (0.5% final DMSO in H226 and HCC827, 1% final DMSO in all other cell lines) was added to cells in separate wells as a control. Three days thereafter, cellular proliferation was measured by AlamarBlue and normalized to the solvent control.
Regression trendlines & IC50 values: Linear and non-linear regression trendlines were generated using the CompuSyn (ComboSyn, Inc, Paramus, N.J.) and Graphpad (Prism) software, respectively. The non-linear trendlines provided a better fit for the actual data and were used to calculate IC50, IC25 and other drug concentrations (ICx), although both software programs generated similar values.
Combination Effects Determined by the “Fixed Concentration” Method
The “Fixed Concentration” method was used for cell lines with acquired resistance (HCC827res). Cells were reverse-transfected with miR-34a using the miRNA at a fixed, weak concentration (˜IC25) as described above. The following day, cells were treated with erlotinib in a serial dilution (0.01-100 μM). Cell proliferation inhibition was analyzed 3 days later by AlamarBlue. To measure the effects of the single agents and to correct for effects potentially contributed by lipid carrier or vehicle, cells were also treated with miR-34a in combination with solvent (0.5% DMSO in HCC827res, 1% DMSO in all other cell lines) or erlotinib in combination with mock-transfection. All proliferation data was normalized to mock-transfected cells treated with solvent (DMSO). The combinatorial effect was evaluated by a visual inspection of the erlotinib dose-response curve and a shift of the IC50 value in the presence or absence of miR-34a (graphed and calculated using Graphpad).
Combination Effects Determined by the “Fixed Ratio” Method
Cells were treated with 7 concentrations of erlotinib each in combination with 7 concentrations of miR-34a. Each drug was used at a concentration approximately equal to its IC50 and at concentrations within 2.5-fold (NSCLC) or 2-fold (HCC) increments above or below. This matrix yielded a total of 49 different combinations representing 13 different ratios. Each drug was also used alone at these concentrations. miR-34a and erlotinib were added as described above, and cellular proliferation was determined by AlamarBlue. Each data point was performed in triplicates.
Calculation of Combination Index (CI) Values
CI values based on Loewe's additivity model were determined to assess the nature of drug-drug interactions that can be additive (CI=1), antagonistic (CI>1), or synergistic (CI<1) for various drug-drug concentrations and effect levels (Fa, fraction affected; inhibition of cancer cell proliferation). Both linear regression and nonlinear regression trendlines were used to calculate and compare CI values. CI values based on linear regression analysis was done using the CompuSyn software (ComboSyn Inc., Paramus, N.J.), following the method by Chou et al., whereby the hyperbolic and sigmoidal dose-effect curves are transformed into a linear form (Chou T C (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70: 440-6, instructions also available at ComboSyn, Inc., www.combosyn.com). CI values derived from non-linear regression trendlines were calculated using Equation 1 in which CA,x and CB,x are the concentrations of drug A and drug B in the combination to produce effect X (Fa). ICx,A and ICx,B are the concentrations of drug A and drug B used as a single agent to produce that same effect.
Drug concentrations required in Equation 1 to determine CI values (CA,x, CB,x, ICx,A and ICx,B) were calculated using the Hill equation (Equation 2), IC50 and Hill slope value (n) derived from non-linear regression trendlines (Graphpad).
Isobolograms
To describe the dose-dependent interaction of erlotinib and miR-34a, isobolograms at effect levels of 50% and 80% inhibition of cancer cell proliferation were created. Since the single agents—alone or in combination—usually reached 50% cancer cell inhibition, the 50% isobologram provided an actual comparison of the single use vs. the combination. The 80% isobologram was used to illustrate the utility of the combination at a high effect level that have practical implications in oncology. In each of these, additivity was determined by extrapolating the dose requirements for each drug in combination from its single use (IC50, IC80). Data points above or below the line of additivity indicate antagonism or synergy, respectively. For all 49 combinations, drug concentrations required in the combination were compared to those of the single agents alone to reach the same effect and expressed as a fold change (dose reduction index, DRI).
Curve Shift Analysis
To allow a direct comparison of the dose-response curves and to identify synergistic drug-drug interaction, non-linear regression trendlines of each drug alone or of the combination (IC50:IC50 ratio or other ratios where indicated) were normalized to its own IC50 value and referred to as IC50 equivalents (IC50 eq). IC50 equivalents of the combination were calculated using Equation 3 and described in Zhao L, Au J L Wientjes M G (2010) Comparison of methods for evaluating drug-drug interaction. Front Biosci (Elite Ed) 2: 241-9. Data of the single agents and in combination were graphed in the same diagram to illustrate lower drug concentrations required to achieve any given effect relative to the single agents. This is represented in a left-shift of the dose-response curve and indicates synergy. Id.
Statistical Analysis
Statistical analysis was done using the Excel (Microsoft), CompuSyn and Graphpad software. Averages and standard deviations were calculated from triplicate experiments. Goodness of fit of linear and non-linear regression trendlines was described by R (CompuSyn) and R2 (Graphpad) values, respectively, and were >0.9 for most cell lines except H226 and HepG2 cells due to limiting drug insensitivity.
Results
miR-34a Restores Sensitivity to Erlotinib in Non-Small Cell Lung Cancer Cells
To study drug resistance in cells with acquired resistance, we used HCC827 cells that express an activating EGFR mutation (deletion of exon 19 resulting in deletion of amino acids 745-750). HCC827 are highly sensitive to erlotinib with an IC50 value of 0.022 μM (
Since both MET and AXL are directly repressed by miR-34, and because inhibition of AXL can antagonize erlotinib resistance, the introduction of synthetic miR-34 mimics may restore erlotinib sensitivity. To explore this possibility, HCC827res cells were exposed to increasing erlotinib concentrations, ranging from 0.03-100 μM, either in the absence or presence of miR-34a used at a fixed, weak concentration (0.3 nM). The effects of erlotinib were expected to be concentration-dependent, such that erlotinib in combination with miR-34a produced lower IC50 values relative to erlotinib alone. As shown in
To determine whether the miRNA can also counteract primary resistance mechanisms, we used H1299 cells that have mutations in the NRAS and TP53 genes. In these cells, erlotinib produced an IC50 value of 11.0 μM (
miR-34a and Erlotinib Synergize in Non-Small Cell Lung Cancer Cells
The shift of the erlotinib IC50 value demonstrated how a fixed miR-34a concentration can improve the potency of erlotinib. However, this model, also known as “Fixed-Concentration-Model”, does not allow the assessment of synergy. To investigate whether both drugs can enhance each other, we employed the “Fixed-Ratio-Model” that is based on Loewe's concept of additivity (Chou T C (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70: 440-6. Tallarida R J (2001) Drug synergism: its detection and applications. J Pharmacol Exp Ther 298: 865-72. Tallarida R J (2006) An overview of drug combination analysis with isobolograms. J Pharmacol Exp Ther 319: 1-7.) In this model, combination index (CI) values are calculated based on the slope and IC50 value of each dose-response curve (drug alone or in combination) and define whether the drug-drug interactions are synergistic (CI<1), additive (CI=1), or antagonistic (CI>1). Since the accuracy of the CI values depends on the fit of the dose-response curve trendline, CI values were calculated by two methods using either linear or non-linear regression trendlines (see Materials and Methods). Four erlotinib-resistant cell lines were used, all of which differ in their genetic make-up: A549 (mutations in KRAS, STK11, CDKN2A), H460 (mutations in KRAS, STK11, CDKN2A, PIK3CA), H1299 (mutations in NRAS, TP53), and H226 (mutations in CDKN2A) [37]. A qRT-PCR analysis showed a marked increase of AXL, GAS6 and FGFR1 mRNA levels in these cells relative to erlotinib-sensitive HCC827 cells, further providing an explanation for erlotinib resistance (
Next, we generated isobolograms and determined the dose requirements for each drug at 50% and 80% cancer cell inhibition as a read-out for synergy. The 50% effect level was chosen because the potency of a drug is frequently assessed at its IC50 and because in our studies each drug alone was capable of inhibiting most cancer cells by 50%, allowing a comparison of each drug alone with the combination within the range of actual data. The 80% effect level was chosen because it is important to demonstrate synergy at high inhibitory activity for oncology applications. Although the concentrations of each drug alone to achieve 80% inhibition are based on an extrapolation of the dose-response curve and are theoretical in nature, the miR-34a-erlotinib combination readily achieved 80% inhibition or greater and is within the range of actual data. Since the two drugs by themselves were not very effective in H226 cells, isobolograms at 30% and 50% inhibition were created for H226 data. As shown in
Third, we performed curve-shift analyses whereby the concentration of each drug has been normalized to its own IC50 value (Zhao L, Au J L Wientjes M G (2010) Comparison of methods for evaluating drug-drug interaction. Front Biosci (Elite Ed) 2: 241-9.). This conversion of drug concentrations into IC50 equivalents (IC50 eq) allows a direct comparison of each dose-response curve from the single agents and the combination. Trendlines were generated and span effect levels from 0-100% inhibition. The slope of the trendline indicates drug potency, and the maximal activity can be guaged from actual data points. Synergy is identified when IC50 equivalents of the combination are lower to achieve any given effect relative to the single agents. Id. This is visually indicated by a left-shift of the combination trendline. As seen in
Various Ratios of Erlotinib and miR-34a Cooperate Synergistically
Our analysis suggests that erlotinib and miR-34a synergize when the two drugs are combined at a ratio derived from their IC50 values. Because drug-drug interactions can change depending on the relative amounts, we explored the effects of multiple erlotinib-miR-34a ratios by combining erlotinib at concentrations from 0.41-100 μM with miR-34a at concentrations from 0.12-30 nM. Drug doses were increased in 2.5-fold increments, and each drug was also used alone as controls. This matrix yielded 49 drug combinations representing 13 different drug ratios (
Calculated CI values predict that erlotinib and miR-34a combined at all of these ratios provide strong synergy (
The full range of 49 combinations was also tested in H1299, H460 and H226 cells and confirmed the results obtained with A549 cells (
Erlotinib and miR-34a Cooperate Synergistically in Hepatocellular Carcinoma Cells
To investigate whether the cooperative activity of erlotinib and miR-34a has utility in other cancer indications, we probed this combination in cell models of hepatocellular carcinoma. Liver cancer was chosen as test platform because erlotinib is moderately effective in patients with advanced liver as a single agent and failed to prolong overall survival and time-to-progression in combination with sorafenib (Philip P A, Mahoney M R, Allmer C, Thomas J, Pitot H C, et al. (2005) Phase II study of Erlotinib (OSI-774) in patients with advanced hepatocellular cancer. J Clin Oncol 23: 6657-63. Thomas M B, Chadha R, Glover K, Wang X, Morris J, et al. (2007) Phase 2 study of erlotinib in patients with unresectable hepatocellular carcinoma. Cancer 110: 1059-67. Zhu A X, Rosmorduc O, Evans J, Ross P, Santoro A, et al. (2012) SEARCH: A phase III, randomized, double-blind, placebo-controlled trial of sorafenib plus erlotinib in patients with hepatocellular carcinoma (HCC). 37th Annual European Society for Medical Oncology Congress, Vienna, Austria, September 28-October 2 (abstr 917)).
In addition, MRX34, a miR-34a liposome currently in clinical testing, effectively eliminated liver tumors in preclinical animal studies and therefore may be an attractive agent in combination with erlotinib. Cell models used included Hep3B, C3A, HepG2 and Huh7, several of which showed an upregulation of erlotinib-resistance genes, AXL, HGF, FGFR1 and ERBB3 in comparison to an erlotinib-sensitive lung cancer line (
Discussion
An accurate evaluation of drug-drug interactions is complex because outcomes depend on drug ratios, drug concentrations and desired potency (Chou T C (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70: 440-6). To investigate the pharmacological relationship between miR-34a mimics and erlotinib, we used multiple analytical approaches to reveal drug enhancements (“Fixed Concentration” model) and to distinguish between additivity, antagonism and synergy (“Fixed Ratio” model). We examined CI values, isobolograms and IC50 equivalents derived from linear or non-linear data regression. Our data show that miR-34a augments the sensitivity to erlotinib in all cancer cells tested—whether they were associated with primary or secondary/acquired resistance. A plausible explanation is provided by the fact that tumor suppressor miRNAs inhibit numerous cancer pathways. In support of this hypothesis, AXL and MET, gene products specifically linked to erlotinib resistance, are directly repressed by miR-34a (Kaller M, Liffers S T, Oeljeklaus S, Kuhlmann K, Roh S, et al. (2011) Genome-wide characterization of miR-34a induced changes in protein and mRNA expression by a combined pulsed SILAC and microarray analysis. Mol Cell Proteomics 10: M111 010462. Mudduluru G, Ceppi P, Kumarswamy R, Scagliotti G V, Papotti M, et al. (2011) Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene 30: 2888-99. He L, He X, Lim L P, de Stanchina E, Xuan Z, et al. (2007) A microRNA component of the p53 tumour suppressor network. Nature 447: 1130-4.).
Unexpectedly, erlotinib also enhanced the therapeutic effects of the miR-34a mimic, despite existing evidence implicating miR-34a in the control of multiple oncogenic signaling pathways, including the EGFR pathway (Lal A, Thomas M P, Altschuler G, Navarro F, O'Day E, et al. (2011) Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet 7: e1002363.). Thus, this result demonstrates that a miRNA mimic can synergize with a single gene-directed therapy and invites the search for other combinations. Accordingly, in various additional embodiments, the present invention includes combinations of miR-34a with other EGFR inhibitors, such as gefitinib, afatinib, panitumumab and cetuximab, as well as HER2 inhibitors such as lapatinib, pertuzumab and trastuzumab.
In lung cancer cells with acquired resistance (HCC827res), adding a small amount of miR-34a was capable of reducing erlotinib IC50 values below 0.1 μM. This is a remarkable result and suggests that miR-34a can render this cell line equally erlotinib-sensitive compared to parental HCC827 cells. In lung cancer cells with primary resistance, the IC50 dose requirement for erlotinib decreased by 4- to 46-fold and was approximately 2 μM. This may be within the range of concentrations that have clinical utility (Sharma S V, Bell D W, Settleman J Haber D A (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7: 169-81.). Erlotinib is given as a daily, oral dose of up to 150 mg. Although the clinical dose level of MRX34 has yet to be established, the molar ratios between miR-34a and erlotinib used in the clinic are likely within the range of ratios that have shown synergy in our cell studies.
Erlotinib is currently used as a first-line therapy for NSCLC patients with activating EGFR mutations. It is also used as a maintenance therapy after chemotherapy and second- and third-line therapy for locally advanced or metastatic NSCLC that has failed at least one prior chemotherapy regimen. Clinical trials failed to demonstrate a survival benefit of erlotinib in combination with cisplatin/gemcitabine or carboplatin/paclitaxel compared to conventional chemotherapies alone (Id. Herbst R S, Prager D, Hermann R, Fehrenbacher L, Johnson B E, et al. (2005) TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 23: 5892-9.). A recent Phase III trial, investigating erlotinib plus sorafenib in HCC, also did not meet its endpoint (Zhu A X, Rosmorduc O, Evans J, Ross P, Santoro A, et al. (2012) SEARCH: A phase III, randomized, double-blind, placebo-controlled trial of sorafenib plus erlotinib in patients with hepatocellular carcinoma (HCC). 37th Annual European Society for Medical Oncology Congress, Vienna, Austria, September 28-October 2 (abstr 917)). Thus, other approaches for combination therapies are desired. Our data show that the erlotinib plus miR-34a combination is particularly effective and may substantially broaden the NSCLC patient population that can be treated with erlotinib. The combination was similarly synergistic in HCC cells, suggesting that the synergistic interaction is a result of their molecular mechanisms of action and can also be applied to cancers other than NSCLC.
The human breast cancer cell lines BT-549, T47D, MDA-MD-231 and MCF-7 (from ATCC) were used to evaluate the combinatorial effects of mir-Rx34 and lapatinib. Lapatinib was purchased from LC Laboratories (Woburn, Mass.). Synthetic miR-34a and miR-NC mimics were manufactured by Life Technologies (Ambion, Austin, Tex.). To determine the IC50 value of each drug alone, 2,000-3,500 cells per well were seeded in a 96-well plate format and treated with either lapatinib or miR-34a as follows. (i) miR-34a mimics were reverse-transfected in triplicates in a serial dilution (0.03-30 nM) using RNAiMax lipofectamine from Invitrogen according to a published protocol. As controls, cells were also transfected with RNAiMax alone (mock). Cells were incubated with AlamarBlue (Invitrogen) 6 days post transfection to determine cellular proliferation. Proliferation data were normalized to mock-transfected cells. (ii) Lapatinib, prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO), was added to cells one day after seeding at a final concentration ranging from 0.1 and 100 μM. Solvent alone (1% final DMSO in all cell lines) was added to cells in separate wells as a control. Three days thereafter, cellular proliferation was measured by AlamarBlue and normalized to the solvent control.
The combination studies were carried out at ˜IC50 ratio of lapatinib and miR-Rx34 (ratio=IC50 lapatinib/IC50 miR-Rx34). Cells were treated with lapatinib in combination with miR-Rx34a at a concentration approximately equal to its corresponding IC50 and concentrations within 2 fold increments above or below. The ratios of lapatinib/miR-Rx34a are 4000 in BT-549, 3333.3 in MDA-MD-231, 5000 in MCF-7 and 6000 in T47D. Cells were reversed transfected with miR-Rx34a, lapatinib were added 3 days post transfection, and cell proliferation were measured 3 days post lapatinib addition by AlamarBlue.
CI values were calculated based on non-linear regression of dose-response curves of the single agents and when used in combination, and are shown relative to the level of cancer cell inhibition on an axis from 0 (no inhibition) to 1 (100% inhibition). Combinations that are considered synergistic and have clinical value are those with a low CI value (<0.6) at maximal cancer cell inhibition. As shown in
To treat patients with non-small cell lung cancer, a MRX34+erlotinib combination can be used as follows. Patient is given a daily oral dose of 150, 100, or 50 mg erlotinib and an intravenous 30 min to 3 hr infusion of MRX34 at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124, or 165 mg/m2.
In another example erlotinib is given as a daily oral dose of 150, 100, or 50 mg and MRX34 is given three twice a week (for instance Mondays and Thursdays) during a 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example, erlotinib is given as a daily oral dose of 150, 100, or 50 mg and MRX34 is given daily by an intravenous 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2 on five consecutive days with the following two days off per week. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
To treat patients with pancreatic cancer, for example pancreatic ductal adenocarcinoma, a MRX34+erlotinib combination can be used as follows. Patient is given a daily oral dose of 100 or 50 mg erlotinib and an intravenous 30 min to 3 hr infusion of MRX34 at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example erlotinib is given as a daily oral dose of 100 or 50 mg, and MRX34 is given three twice a week (for instance Mondays and Thursdays) during a 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example, erlotinib is given as a daily oral dose of 100 or 50 mg, and MRX34 is given daily by an intravenous 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2 on five consecutive days with the following two days off per week. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
To treat patients with breast cancer, for example hormone receptor-positive, HER2-positive metastatic breast cancer, a MRX34+lapatinib combination can be used as follows. Patient is given a daily oral dose of 1500, 1250, 1000, or 750 mg lapatinib and an intravenous 30 min to 3 hr infusion of MRX34 at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example lapatinib is given as a daily oral dose of 1500, 1250, 1000, or 750 mg, and MRX34 is given three twice a week (for instance Mondays and Thursdays) during a 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example, lapatinib is given as a daily oral dose of 1500, 1250, 1000, or 750 mg, and MRX34 is given daily by an intravenous 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2 on five consecutive days with the following two days off per week. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example, lapatinib and MRX34 is given as described above and combined with capecitabine 2,000 mg/m2/day (administered orally in 2 doses approximately 12 hours apart) on Days 1-14 in a repeating 21-day cycle.
In another example, lapatinib and MRX34 are given as described above and combined with letrozole 2.5 mg once daily
To treat patients with non-small cell lung cancer, a MRX34+afatinib combination can be used as follows. Patient is given a daily oral dose of 40, 30, or 20 mg afatinib and an intravenous 30 min to 3 hr infusion of MRX34 at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example afatinib is given as a daily oral dose of 40, 30, or 20 mg, and MRX34 is given three twice a week (for instance Mondays and Thursdays) during a 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
In another example, afatinib is given as a daily oral dose of 40, 30, or 20 mg, and MRX34 is given daily by an intravenous 30 min to 3 hr infusion at dose levels ranging from 50 mg/m2 to 165 mg/m2 on five consecutive days with the following two days off per week. In particular situations, MRX34 is given at dose levels of 50, 70, 93, 124 or 165 mg/m2.
The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims benefit of priority to U.S. Ser. No. 61/787,558, filed Mar. 15, 2013 and U.S. Ser. No. 61/927,543, filed Jan. 15, 2014, which are both incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61927543 | Jan 2014 | US | |
| 61787558 | Mar 2013 | US |
