DEPLETING EGFR AND HER2 OVERCOMES RESISTANCE TO EGFR INHIBITORS IN COLORECTAL CANCER

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “003551_01063_ST26.xml”, was created on Nov. 15, 2022, and is 14,909 bytes in size.

BACKGROUND OF THE DISCLOSURE

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is a well-known oncogenic driver and a therapeutic target in several types of human cancer. It functions by forming homodimeric and heterodimeric signaling units, which activate various oncogenic signaling pathways in cancer cells. More than 80% of primary and metastatic colorectal cancers (CRCs) are EGFR-positive, with overexpression in about 60% and gene amplification in about 10% of the cases [1, 2]. Two classes of EGFR inhibitors are available clinically, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (MABs). However, EGFR TKIs have not shown significant therapeutic activity in CRC [3]. Two EGFR-directed MABs are approved for treatment of patients with CRC, including cetuximab and panitumumab, but only 10-20% of the patients respond to treatment and response lasts typically 3-12 months [4, 5]. The two MABs show similar efficacy in CRC patients [6]. The molecular basis underlying response to the MABs is not well known. Many mechanisms that confer primary or acquired resistance to the EGFR inhibitors in CRC have been reported, including but not limited to activating mutations of KRAS, NRAS, BRAF, and PIK3CA [7, 8]. Most notably, activating KRAS mutations occur in up to nearly half of metastatic CRC cases [9, 10], and this group of patients do not benefit from the EGFR MABs [11, 12]. It is widely believed that CRC cells are rendered independent of EGFR by changes in other signaling molecules. However, response to therapeutic targeting of KRAS, BRAF or PIK3CA is very limited if any [13-18]. Therefore, there is an ongoing and unmet need to provide alternative approaches to treating CRC, and other types of EGFR positive cancers. The present disclosure is pertinent to this need.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for treating cancer. In arriving at the described methods, we compared the response of a panel of CRC cell lines to cetuximab, panitumumab, and PEPD^G278D. PEPD^G278Dis a recombinant and enzymatically inactive mutant of human peptidase D (PEPD), with replacement of glycine 278 by aspartic acid. We recently showed that PEPD^G278Dinduces the internalization and degradation of both EGFR and its family member HER2 by binding directly to their extracellular domains [19-21, the disclosures of which are incorporated herein by reference]. We also showed that PEPD^G278Dinactivates other RTKS indirectly by disrupting their heterodimerization with EGFR or HER2 [21, 22, the disclosures of which are incorporated herein by reference]. Consequently, PEPD^G278Dstrongly inhibits the growth of cancer cells and tumors overexpressing EGFR and/or HER2 [19-22]. EGFR and HER2 appear to be the only direct targets of PEPD^G278D, as we showed that cells and tumors lacking these RTKs do not respond to PEPD^G278D[19-22]. The CRC cell lines used in this disclosure express EGFR and HER2 at different levels or have no expression of these RTKs. Some of the cell lines harbor activating mutations of KRAS, BRAF and/or PIK3CA. siRNA knockdown of EGFR or HER2 was carried out to assist data interpretation. We also evaluated the effect of PEPD^G278Don EGFR mutants which occur in patient CRC and are insensitive to cetuximab or panitumumab. Moreover, we compared the inhibitory activities of cetuximab and PEPD^G278Din vivo using mouse CRC models with and without mutations of KRAS, BRAF and/or PIK3CA.

The results show unexpectedly that CRC resistance to cetuximab and panitumumab results primarily from their inability to downregulate EGFR. Presence of activating mutations of KRAS, BRAF and/or PIK3CA does not curtail inhibition of oncogenic signaling and cell growth induced by PEPD^G278Dvia depletion of EGFR and HER2. siRNA knockdown of EGFR or HER2 also inhibits the growth of CRC cells resistant to the EGFR MABs. PEPD^G278Dtargets wild type (WT) and mutated EGFR with similar efficacy. In CRC tumor models resistant to the EGFR MABs, tumor-generated high affinity EGFR ligands compete with PEPD^G278Dfor EGFR binding, but aderbasib, an inhibitor of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and ADAM17 [23], inhibits EGFR ligand shedding from tumor cells and allows target engagement by PEPD^G278D, resulting in significant inhibition of tumor growth, while aderbasib as a monotherapy is inactive. ADAM10 and ADAM17 are responsible for shedding of all EGFR ligands from cells [24, 25]. Moreover, the antitumor activity of PEPD^G278Dis further enhanced by fluorouracil (5-FU), which is widely used in CRC treatment. Collectively, the present results show that CRC resistance to EGFR inhibitors stems primarily from the inability of the inhibitors to downregulate EGFR, rather than mutations in EGFR, KRAS, BRAF and PIK3CA, and support a therapeutic strategy centered on the use of PEPD^G278Dand related PEPD proteins for overcoming drug resistance in CRC and other cancers. Thus, the disclosure provides in certain embodiments for administering to an individual in need thereof a combination of PEPD and an inhibitor of one or more a sheddases, such as ADAM10 and ADAM17. The disclosure further includes administering to the individual a chemotherapeutic agent, such as 5-FU to enhance anti-cancer activity. The method further comprises administering to the individual a coagulation inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

FIG. 1. PEPD^G278Dinhibits CRC cells resistant to cetuximab. (A) Western blotting of untreated whole cell lysates. (B) Effects of PEPD^G278Dand cetuximab on cell growth measured by MTT assay. Each value is mean±SD (n=3). ****P<0.0001 by one-way ANOVA, followed by Tukey test for comparison with the control. (C) Western blotting of whole cell lysates after treatment of the cells with vehicle, PEPD^G278D(25 nM), or cetuximab (275 nM) for 48 h. The following phosphorylation sites were measured: pY1173-EGFR, pY1221/1222-HER2, pY1328-HER3, pY1234/1235-MET, pY1131-IGF1R, pY416-SRC, pS473-AKT, and pT202/Y204-ERK. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured as a loading control here and elsewhere. HCT116 cell lysates were used as a positive control for measurement of p-SRC and p-AKT in SW48 cells and for measurement of EGFR and HER2 in SW620 cells.

FIG. 2. PEPD^G278Dinhibits RAS and PI3K and targets EGFR mutants in CRC cells. (A) Western blotting of whole cell lysates from cells treated with solvent, PEPD^G278D(25 nM), and cetuximab (275 nM) for 48 h. (B), (C) Ras and PI3K activities in whole cell lysates from cells treated with solvent or PEPD^G278D(25 nM) for 48 h. Each value is mean±SD (n=3). *P<0.05, ****P<0.0001 by two-tailed unpaired t test. (D) Western blotting of whole cell lysates from untreated cells or cells which were transfected with EGFR or its mutant and 24 h later treated with solvent or PEPD^G278D(25 nM) for 48 h. p-MEK and p-EGFR/mutant are pS217/221-MEK and pY1173-EGFR, respectively.

FIG. 3. PEPD^G278Dinduction of EGFR degradation is slowed by EGFR ligands. (A) Western blotting of whole cell lysates from untreated cells, cells treated with PEPD^G278D(25 nM) for different times in medium containing 10% FBS, no FBS, or HB-EGF (20 ng/ml) without FBS, or from cells treated with HB-EGF (20 ng/ml) without PEPD^G278Dand FBS. (B) Confocal fluorescence staining of EGFR, PEPD^G278Dand nuclei (DAPI) in SW48 cells treated with PEPD^G278D(25 nM) for 15 min or 6 h. (C) Confocal fluorescence staining of EGFR, LMAP1 and nuclei (DAPI) in SW48 cells with no treatment, or treated with PEPD^G278D(25 nM) and/or chloroquine (25 μM) for 1 or 6 h. Scale bars in (B), (C): 10 Qim.

FIG. 4. The antitumor activities of cetuximab and PEPD^G278Din vivo correlate with EGFR downregulation. (A) Mice bearing subcutaneous tumors were treated with EP daily (SW48 tumors: days 2-20; HCT116 tumors: days 3-20; HT29 tumors: days 8-24; n=12), cetuximab twice weekly (SW48 tumors: days 8-20; HT116 tumors: days 7-20; HT29 tumors: 10-24; n=12-14), or EP daily plus PEPD^G278Dthrice weekly (SW48 tumors: days 8-20; HCT116 tumors: days 7-20; HT29 tumors: days 10-24; n=12-14). EP, cetuximab, and PEPD^G278Dwere administered ip at 0.5, 15, and 4 mg/kg per dose, respectively. Group average tumor sizes were 107-111 mm³(SW48 tumors), 100-129 mm³(HCT116 tumors), and 219-229 mm³(HT29 tumors) at the beginning of treatment. Each value is mean±SEM. ****P<0.0001 by one-way ANOVA, followed by Tukey test for comparison with the control. (B) Western blotting of tumor homogenates (2 tumors per group). Tumors were harvested 24 h after the final treatment. See FIG. 1 legend for protein phosphorylation sites. (C) Plasma levels of mouse PEPD and PEPD^G278Dat 24 h after final PEPD^G278Dtreatment. Each value is mean±SD (n=3). n.d., not significant by two-tailed unpaired t test. (D), (E) Levels of soluble AREG and HB-EGF in control tumors. Each value is mean±SD (n=3). ***P<0.001, ****P<0.0001, by two-tailed unpaired t test.

FIG. 5. Aderbasib restores the antitumor activity of PEPD^G278Dby inhibiting HB-EGF shedding. (A) Mice bearing subcutaneous tumors were randomized to EP treatment (n=12-16), aderbasib treatment (n=12), or treatment with EP plus aderbasib and PEPD^G278D(n=12). Mice were treated with EP daily (HCT116 tumors: days 2-17; HT29 tumors: days 3-17), aderbasib daily (HCT116 tumors: days 4-17; HT29 tumors: days 4-17), and PEPD^G278Dthrice weekly (HCT116 tumors: days 5-17; HT29 tumors: days 5-17). EP and PEPD^G278Dwere administered to mice by ip at 0.5, and 4 mg/kg per dose, respectively. Aderbasib was administered to mice by gavage at 60 mg/kg per dose. Group average tumor sizes were 92-110 mm³(HCT116 tumors) and 153-172 mm³(HT29 tumors) at the beginning of treatment. Each value is mean±SEM. **P<0.01, ****P<0.001, by one-way ANOVA, followed by Tukey test. (B) Tumor levels of soluble HB-EGF. Each value is mean±SD (n=3). ****P<0.0001 by one-way ANOVA, followed by Tukey test. c Western blotting of tumor homogenates (2 tumors per group). Tumors were harvested 24 h after final treatment. See FIG. 1 and FIG. 2 legends for protein phosphorylation sites.

FIG. 6. 5-FU enhances the therapeutic outcome of the PEPD^G278D-centered combination treatment. (A)-(C) Mice bearing subcutaneous tumors were treated with EP daily (HCT116 tumors: days 3-20; HT29 tumors: days 3-17; PDX14650: days 4-23), or EP daily plus aderbasib daily (HCT116 tumors: days 4-20; HT29 tumors: days 4-17; PDX14650: days 6-23) plus PEPD^G278Dthrice weekly (HCT116 tumors: days 5-19; HT29 tumors: days 5-17; PDX14650: days 7-23) plus 5-FU every 3-4 days (HCT116 tumors: days 6-20; HT29 tumors: days 6-17; PDX14650: days 8-23). EP, PEPD^G278D, and 5-FU were administered to mice by ip at 0.5, 4 and 35 mg/kg per dose, respectively. Aderbasib was administered to mice by gavage at 60 mg/kg. Group average tumor volumes were 59-64 mm³(HCT116 tumors), 153-185 mm³(HT29 tumors), and 193-226 mm³(PDX14650) at the beginning of treatment. Each value is mean±SEM (n=13-16). ****P<0.0001 by two-tailed unpaired t test. (D) Western blotting of tumor homogenates (2 tumors per group). Tumors were harvested 24 or 48 h after final PEPD^G278Dtreatment. See FIG. 1 and FIG. 2 legends for protein phosphorylation sites.

FIG. 7. 5-FU plus PEPD^G278D-based combination treatment inhibits orthotopic CRC. (A) Tumor burden measured by bioluminescence imaging. Tumor-bearing mice were treated by EP daily (days 22-56), or EP daily plus aderbasib daily (days 26-56) plus PEPD^G278Dthrice weekly (days 27-55) plus 5-FU every 3-4 days (days 28-56). EP, PEPD^G278Dand 5-FU were administered to mice by ip at 0.5, 4 and 35 mg/kg per dose, respectively. Aderbasib was administered to mice by gavage at 60 mg/kg. Each value is mean±SEM (n=11-12). **P<0.01 by Mann-Whitney U test. (B)-(D) Percentage of mice showing frank primary tumors, average tumor weight, and percentage of mice showing liver metastasis at the end of experiment. e Western blotting of tumor homogenates (2 tumors per group). Tumors were harvested on day 57. See FIG. 1 and FIG. 2 legends for protein phosphorylation sites.

FIG. 8. KRAS, BRAF and/or PIK3CA are mutated in two CRC cell lines and PDX14650. (A) Gene mutation analysis in cell lines, including KRAS, BRAF, and PIK3CA. (B) Analysis of KRAS mutation in PDX14650.

FIG. 9. The growth-inhibitory activity of panitumumab in CRC cell lines correlates with EGFR downregulation. (A) Effects of panitumumab on cell growth measured by MTT assay. Each value is mean±SD (n=3). ****P<0.0001 by one-way ANOVA, followed by Tukey test for comparison with the control. (B) Western blotting of whole cell lysates after treatment of the cells with vehicle or panitumumab (277 nM) for 48 h.

FIG. 10. PEPD^G278Ddisrupts EGFR-HER3, EGFR-IGF1R and EGFR-MET heterodimers. Western blotting of anti-HER3 IP, anti-IGF1R IP or anti-MET IP of whole cell lysates from cells treated with PEPD^G278D(25 nM) for 48 h.

FIG. 11. CRC cells resistant to EGFR MABs require EGFR and HER2. (A) Western blotting of whole cell lysates after treatment of cells with EGFR siRNA or HER2 siRNA for 48 h. (B) Effects of knockdown of EGFR or HER2 by siRNA on cell growth measured by MTT assay. Each value is mean±SD (n=3). ****P<0.0001 by two-tailed unpaired t test.

FIG. 12. Cetuximab attenuates cell growth inhibition by PEPD^G278Din vitro, and EP plus high dose of PEPD^G278Ddo not inhibit tumor growth in vivo. (A) Effect of combining PEPD^G278Dwith cetuximab on cell growth measured by MTT assay. Each value is mean±SD (n=3). ****P<0.0001 by two-tailed unpaired t test. (B) Mice bearing subcutaneous HCT116 tumors were randomized to EP, or EP plus PEPD^G278D. EP was administered to mice at 0.5 mg/kg per dose daily by ip (days 3-24). PEPD^G278Dwas administered to mice at 8 mg/kg per dose thrice weekly by ip (days 10-24). Each value is mean±SEM (n=6).

FIG. 13. Aderbasib restores the antitumor activity of PEPD^G278Din a dose-dependent manner. (A)-(C) Mice bearing subcutaneous tumors were randomized to EP (n=12-16), aderbasib (n=12), or EP plus aderbasib plus PEPD^G278D(n=12). EP was administered to mice daily by ip at 0.5 mg/kg per dose (HCT116 tumors in (A): days 3-20; HCT116 tumors in (B): days 2-16; HT29 tumors in c: days 2-19). Aderbasib was administered to mice daily by gavage at 15 or 30 mg/kg per dose (HCT116 tumors in (A): days 4-20; HCT116 tumors in (A): days 4-16; HT29 tumors in (C): days 4-19). PEPD^G278Dwas administered to mice thrice weekly by ip at 4 mg/kg per dose (HCT116 tumors in (A): days 5-20; HCT116 tumors in (B): days 5-16; HT29 tumors in (C): days 5-19). Each value is mean±SEM. *P<0.01, ***P<0.001, n.d., not different, by one-way ANOVA, followed by Tukey test.

FIG. 14. 5-FU alone or in combination with EP and PEPD^G278Dwithout aderbasib does not inhibit tumor growth. (A) Mice bearing subcutaneous HCT116 tumors were randomized to EP (n=6), 5-FU (n=6), or EP plus 5-FU plus PEPD^G278D(n=6). EP was administered to mice daily by ip at 0.5 mg/kg per dose (days 4-24). 5-FU was administered to mice by ip every 3-4 days at 35 mg/kg per dose (days 7-24). PEPD^G278Dwas administered to mice by ip thrice weekly at 4 mg/kg per dose (days 6-24). (B) Mice bearing subcutaneous HT29 tumors were randomized to vehicle (n=14) or 5-FU (n=16). Vehicle and 5-FU at 35 mg/kg per dose were administered to mice by ip every 4 days (days 6 to 18). Each value is mean±SEM. n.d., not different.

FIG. 15. HB-EGF levels in PDX14650 tumors. Tumor samples were from the experiment described in FIG. 6C. Tumor levels of soluble HB-EGF (three tumors per group) were measured by ELISA. Each value is mean±SD. ****P<0.0001 by t-test.

FIG. 16. Supporting data for the experiment shown in FIG. 7. (A) Whole-body bioluminescence imaging of representative mice on day 56. (B) Representative colon, liver, and peritoneal tumors. (C) H & E staining of representative tumors. Scale bar: 100 μm.

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. All proteins described herein include proteins that have from 90.0-99.9% identity across their entire lengths to such proteins. The amino acid or polynucleotide sequence as the case may be associated with each GenBank or other database accession number of this disclosure is incorporated herein by reference as presented in the database on the effective filing date of this application or patent.

The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

The present disclosure relates to use of combination therapies that include PEPD. The amino acid sequence of human prolidase (PEPD) in SEQ ID NO:1 is known in the art. SEQ ID NO:1 and the cDNA sequence encoding it is accessible via GenBank accession no. J04605.1; the amino acid sequence is also provided under GenBank accession number AAA60064. In one illustrative but not limiting embodiment, enzymatically active human PEPD has the sequence of SEQ ID NO:1:

(SEQ ID NO: 1)

MAAATGPSFWLGNETLKVPLALFALNRQRLCERLRKNPAVQAGSI

VVLQGGEETQRYCTDTGVLFLQESFFHWAFGVTEPGCYGVIDVDT

GKSTLFVPRLPASHATWMGKIHSKEHFKEKYAVDDVQYVDEIASV

LTSQKPSVLLTLRGVNTDSGSVCREASFDGISKFEVNNTILHPEI

VESR VFKTDMELEVLRYTNKISSEAHREVMKAVKVGMKEYGLES

LFEHYCYSRGGMRHSSYTCICGSGENSAVLHYGHAGAPNDRTIQN

GDMCLFDMGGEYYSVASDITCSFPRNGKFTADQKAVYEAVLLSSR

AVMGAMKPGDWWPDIDRLADRIHLEELAHMGILSGSVDAMVQAHL

GAVEMPHGLGHFLGIDVHDVGGYPEGVERIDEPGLRSLRTARHLQ

PGMVLTVEPGIYFIDHLLDEALADPARASFLNREVLQRFRGFGGV

RIEEDVVVIDSGIELLTCVPRTVEEIEACMAGCDKAFTPFSGPK

In SEQ ID NO:1, the G at position 278 is bolded and italicized and represents the location of a G278D mutation which renders the PEPD enzymatically inactive. Thus, in embodiments, the PEPD used comprises a change of glycine at position 278 to an amino acid other than aspartic acid. The disclosure includes PEPD which has also been modified by conservative amino acid substitutions that are based generally on relative similarity of R-group substituents. Non-limiting examples of such substitutions include gly or ser for ala; lys for arg; gln or his for asn; glu for asp; ser for cys; asn for gln; asp for glu; ala for gly; asn or gln for his; leu or val for ile; ile or val for leu; arg for lys; leu or tyr for met; thr for ser; tyr for trp; phe for tyr; and ile or leu for val. Thus, a PEPD that comprises any single conservative amino acid substitution, or any combination of conservative amino acid substitution, provided the PEPD retains its described function. PEPD used in embodiments of this disclosure can include modifications that enhance its desirable characteristics, such as the capability to bind to or enter a tumor cell or tumor microenvironment, or to enhance circulation time, bioavailability, stability, or uses related to EGFR-positive cell-targeted killing. In embodiments, the PEPD amino acid sequence is within the context of a larger polypeptide. Thus, fusion proteins comprising PEPD are included in the disclosure. In an embodiment, a PEPD protein can be conjugated to an immunoglobulin (Ig) or a fragment thereof to provide a chimeric PEPD/Ig molecule. In an another embodiment, PEPD can be conjugated to a chemotherapeutic agent.

Each of the described agents that are used in the described combination therapies can be administered to an individual in need thereof using any suitable route. In embodiments the administration is parenteral, intraperitoneal, intrapulmonary, oral, and intra-tumoral. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. In embodiments, the PEPD, the sheddase inhibitor, and the chemotherapeutic agent are administered concurrently or sequentially. In an embodiment, the chemotherapeutic agent interferes with nucleotide synthesis or nucleotide incorporation into DNA, or both. In a non-limiting embodiment the chemotherapeutic agent comprises fluorouracil (5-FU). In embodiments, the sheddase inhibitor inhibits the activity of a membrane-bound enzymes that cleaves an extracellular portion of a transmembrane protein and thereby inhibits release of the soluble ectodomain of the protein from the cell surface. In embodiments, the sheddase inhibitor inhibits one or both of ADAM10 and ADAM17. In embodiments, the sheddase inhibitor is Aderbasib, also known as known as INCB007839. In embodiments, Aderbasib is administered to an individual orally. In embodiments, a coagulation inhibitor is also used.

In one embodiment, the coagulation inhibitor is an agent that inhibits PEPDs degradation in vivo, so as to reduce PEPD dose required by patients. In one embodiment, the coagulation inhibitor inhibits conversion of prothrombin to thrombin, or inhibits the participation of thrombin in clot formation. In an embodiment, the coagulation inhibitor interferes with the clotting related function of the clot-promoting proteins known as factor X and factor II. In embodiments, the coagulation inhibitor binds to and activates antithrombin III, and as a consequence, coagulation factors Xa and IIa are inhibited. In an embodiment, the coagulation inhibitor is heparin, such as an unfractionated heparin preparation, or a low molecular weight form of heparin. In an embodiment, the inhibitor is a direct Xa inhibitor, either oral or non-oral, including but not limited to the drugs sold under the trade names RIVAROXABAN, APIXABAN or EDOXABAN. In an embodiment, the coagulation inhibitor may be an inhibitor of other blood coagulation factors, including but not limited to Factors XII, XI and VII. In embodiments, the low molecular weight heparin or other coagulation inhibitor is administered using any suitable vehicle and route of administration. In an embodiment, the low molecular weight heparin is enoxaparin or a pharmaceutically acceptable salt thereof, such as enoxaparin sodium. In embodiments, the coagulation inhibitor is administered by subcutaneous injection. In one embodiment, the coagulation inhibitor is administered orally. In embodiments, the coagulation inhibitor can be given prior to, concurrent with, or subsequent to the PEPD composition, and may be administered with the same number and timing of the PEPD administration(s), or may be administered according to a schedule that is different than the PEPD administration.

The amount of PEPD, aderbasib, the coagulation inhibitor, and a chemotherapeutic agent to be used in the method can be determined by those skilled in the art, given the benefit of the present disclosure. Thus, in one embodiment, an effective amount of a composition of the invention is administered. An effective amount can be an amount of the composition that inhibits growth of cancer cells in the individual, alleviates disease symptoms associated with the cancer, suppresses a malignant phenotype of cancer cells, inhibits growth of cells overexpressing an EFGR receptor, inhibits metastasis of a primary tumor, and combinations thereof. In embodiments, the individual to whom a composition of the invention is administered has, is suspected of having, or is at risk for development and/or recurrence of an a cancer. In embodiments, the cancer is ErbB1- or ErbB2-positive cancer. In embodiments, the cancer is colorectal cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer, stomach cancer, anal cancer, pancreatic cancer, or uterine cancer. In embodiments, the individual has a cancer that is resistant to another anti-cancer agent. In embodiments, the individual has a cancer that is resistant to a therapeutic antibody.

In embodiments, the individual has a cancer that is resistant to another anti-cancer agent. In embodiments, the individual has a cancer that is resistant to a therapeutic antibody. In embodiments, the individual has a cancer that is resistant to a therapeutic antibody or a combination of therapeutic antibodies that specifically bind to EGFR, non-limiting embodiments of which are cetuximab and panitumumab. In embodiments, the individual has a cancer that is resistant to one or more checkpoint inhibitors. In embodiments, the cancer has a mutation that is not present in non-cancer cells. In embodiments, the cancer comprises a mutation of any one or combination of KRAS, PIK3CA, or BRAF. In embodiments, the mutation is an activating mutation. In a non-limiting embodiment, the KRAS mutation comprises KRAS^{G12C or}KRAS^G12D. In embodiments, the cancer has a mutation in any of Bruton's tyrosine kinase (BTK), EGFR, HER2/NEU, HER3, HER4, MEK, or fibroblast growth factor receptor (FGFR).

In embodiments, administering an effective amount of a combination of described agents to an individual results in one or a combination of loss expression or loss of phosphorylation of EGFR, HER2, or a combination thereof. In embodiments, administering a combination of described agents to an individual results in one or a combination of loss of expression and phosphorylation of EGFR and HER2, loss of phosphorylation but not expression of HER3, IGF1R, MET, SRC, AKT, MEK and ERK, loss of expression of HRAS and NRAS, and activation of caspase 3.

In embodiments, administration of a combination of agents as described herein has an improved anti-cancer effect relative to use of the PEPD, the sheddase inhibitor, or the chemotherapeutic agent, as a mono-therapy (with or without the coagulation inhibitor). In embodiments, use of a combination comprising the PEPD, the sheddase inhibitor and the chemotherapeutic agent has an improved anti-cancer effect relative to use of the PEPD and the sheddase inhibitor as dual therapy (with or without the coagulation inhibitor).

The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any matter.

Example 1

This example provides a description of the materials and methods to generate the results described in the examples that follow Example 1.

Antibodies

The following antibodies were purchased from Cell Signaling: anti-EGFR (Cat #2232), anti-pY1173-EGFR (Cat #4407), anti-HER2 (Cat #2165), anti-pY1221/1222-HER2 (Cat #2243), anti-HER3 (Cat #12708), anti-pY1328-HER3 (Cat #14525), anti-insulin-like growth factor 1 receptor (IGFIR, Cat #9750), anti-pY1131-IGFIR (Cat #3021), anti-MET (Cat #8198), anti-pY1234/1235-MET (Cat #3077), anti-MEK (Cat #4694), anti-pS217/221-MEK (Cat #9154), anti-lysosomal-associated membrane protein 1 (LAMP1, Cat #15665), anti-AKT (Cat #4691), anti-pS473-AKT (Cat #4060), anti-ERK (Cat #9102), anti-pT202/Y204-ERK (Cat #9101), anti-SRC (Cat #2123), anti-pY416-SRC (Cat #6943), anti-cleaved caspase 3 (Cat #9661). Anti-His tag (Cat #MA1-21315), goat-anti-mouse secondary antibody conjugated to Alexa-Fluor 488 (Cat #A-11029), and goat anti-rabbit secondary antibody conjugated to Alexa-Fluor 647 (Cat #A32733) were purchased from Thermo Fisher Scientific. Anti-PEPD (Cat #sc-390042) and anti-NRAS (Cat #sc-31) were purchased from Santa Cruz Biotechnology. Anti-heparin-binding EGF-like growth factor (HB-EGF, Cat #AF-259) and anti-amphiregulin (AREG, Cat #AF-262) were purchased from R&D Systems. Anti-mouse IgG conjugated to horseradish peroxidase (IgG-HRP; Cat #NA931V) and anti-rabbit IgG-HRP (Cat #NA934V) were purchased from GE Healthcare. Anti-PEPD (Cat #ab86507) and anti-KRAS (Cat #WH0003845M1) were purchased from Abcam and Sigma-Aldrich, respectively. Anti-HRAS (Cat #18295-1-AP) and anti-GAPDH (Cat #MAB374) were purchased from Proteintech and Millipore, respectively.

Chemicals, Biochemicals, and Enzymes

Recombinant PEPD^G278Dwas generated in our own lab as previously reported [26]. Briefly, PEPD^G278Dwas synthesized in E coli using pBAD/TOPO-PEPD^G278D-His, purified by nickel chromatography, and concentrated in phosphate-buffered saline (PBS) using Ultracel YM-30 Centricon which was purchased from Millipore (MRCFOR030). Recombinant human HB-EGF (Cat #: 259-HE) was obtained from R&D Systems. Aderbasib was purchased from Medical Isotopes (Cat #17322). The following chemicals were purchased from Sigma-Aldrich: enoxaparin (EP, Cat #1235820), 5-FU (Cat #F6627), BSA (Cat #9048-46-8), paraformaldehyde (Cat #F1635), dimethyl sulfoxide (DMSO; Cat #M81802), chloroquine (Cat #C6628), phenylmethanesulfonyl fluoride (PMSF; 329-98-6), phosphatase inhibitor cocktail 2 (Cat #P5726), phosphatase inhibitor cocktail 3 (Cat #P0044), and methylthiazolyldiphenyl-tetrazolium bromide (MTT, Cat #M2128). Lipofectamine RNAiMAX (Cat #13778075), lipofectamine 3000 (Cat #L3000-008), and ProLong Gold antifade reagent with DAPI (Cat #P36941) were purchased from Thermo Fisher Scientific. A protease inhibitor cocktail (Cat #11-836-153-001) was purchased from Roche Applied Science. D-luciferin was purchased from Gold Biotechnology (Cat #LUCK-1G). G-sepharose beads (Cat #17-6002-35), and Matrigel (Cat #356237) were purchased from GE Healthcare and Corning, respectively. Sodium dodecyl sulfate (SDS) was purchased from Bio-Rad (Cat #161-0301). Cell lysis buffer (10×) was purchased from Cell Signaling (Cat #9803).

Assay Kits

Human Amphiregulin/AREG ELISA Kit PicoKine (Cat #EK0304) and human HB-EGF ELISA Kit (Cat #EK0770) were purchased from Boster Biological. PI3-Kinase Activity ELISA: Pico Kit was purchased from Echelon Biosciences (Cat #K-1000S). Ras Activation ELISA Kit was purchased from Cell Biolabs (Cat #STA-440). BCA Protein Assay Kit (reagent A: Cat #23228; reagent B: Cat #1859078) was purchased from Pierce. RNeasy Mini Kit (Cat #74104), and MinElute PCR Purification Kit (Cat #28004) were purchased from Qiagen. Luminata Classico (Cat #WBLUC0500), and Luminata Cresendo (Cat #WBLUR0100) were purchased from Millipore.

Plasmids

pGL4.51[luc2/CMV/Neo] was purchased from Promega (Cat #E132A). pCMV6-A-EGFR-puro, reported previously [20], was used as a template to generate single point mutations of EGFR, including R451C, K467T, and S492R, using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). The primers were purchased from IDT, including primers for generating EGFR^R451C(forward: 5′-tgaacataacatccttgggattatgctccctcaagg-3 (SEQ ID NO:2); reverse: 5′-ccttgagggagcataatcccaaggatgttatgttca-3′ (SEQ ID NO:3), EGFR^K467T(forward: 5-ggagatgtgataatttcaggaaacacaaatttgtgctatgcaaatacaata-3′ (SEQ ID NO:4); reverse: 5′-tattgtatttgcatagcacaaatttgtgtttcctgaaattatcacatctcc-3′ (SEQ ID NO:5), and EGFR^S492R(forward: 5′-ggtcagaaaaccaaaattataaggaacagaggtgaaaacagc-3′ (SEQ ID NO:6); reverse: 5′-gctgttttcacctctgttccttataattttggttttctgacc-3′ (SEQ ID NO:7)). All constructs were confirmed by DNA sequence analysis.

Genotyping of Cell Lines and PDX

It was previously shown that HCT116 cells carry KRAS (G13D) and PIK3CA (H1047R) mutations and that HT29 cells carry BRAF (V600E) and PIK3CA (P449T) mutations [27]. Colon PDX14650 was found previously to carry KRAS (G12D) mutation. We carried out experiments to confirm the genetic changes, using SW48 cells and SW620 as negative controls. Total RNA was isolated from each cell line and the PDX using a RNeasy Mini Kit, following manufacturer's instruction. RNA (500 ng per sample) was reverse transcribed into complementary DNA using previously described method [21]. The corresponding gene amplicons encompassing KRAS G12 and G13, PIK3CA P449 and H1047, and BRAF V600 were amplified by PCR. The PCR conditions used for all reactions are as follows: 95° C. for 2 min, 35 cycles at 95° C. for 30 sec (denaturation), 64° C. (KRAS G12 and G13, PIK3CA P449) or 54° C. (PIK3CA H1047 and BRAF V600) for 30 sec (annealing), and 72° C. for 30 sec (extension), with the final extension performed at 72° C. for 5 min. The primers were purchased from IDT, including primers for KRAS G12 and G13 (forward: 5′-ccatttcggactgggagcgag-3′ (SEQ ID NO:8); reverse: 5′-gcactgtactcctcttgacctgc-3′ (SEQ ID NO:9)), primers for BRAF V600 (forward: 5′-gcacctacacctcagcagtt-3′ (SEQ ID NO:10); reverse: 5′-gacttctggtgccatccaca-3′ (SEQ ID NO:11), primers for PIK3CA p449 (forward: 5′-cccaggtggaatgaatggct-3′ (SEQ ID NO:12); reverse: 5′-accacactgctgaaccagtc-3′ (SEQ ID NO:13)), and primers for PIK3CA H1047 (forward: 5′-acagcatgccaatctcttca-3′ (SEQ ID NO:14); reverse: 5′-ttgctgtaaattctaatgctgttc-3′ (SEQ ID NO:15)). All PCR reaction products were purified using the MinElute PCR purification Kit, following manufacturer's instruction and were subjected to DNA sequencing. Each of the forward primers except for PIK3CA H1047 was used for the sequencing analyses across the specific amino acid sites on each target gene. The following primer was used for sequencing across PIK3CA H1047: 5′-aatgatgcttggctctgga-3′.

Cell Culture

HCT116 cells (Cat #CCL-247), HT29 cells (Cat #HTB-38), SW48 cells (Cat #CCL-231), and SW620 cells (Cat #CCL-227) were from American Type Culture Collection. HCT116 cells stably expressing firefly luciferase were generated by transfecting HCT116 cells with pGL4.51[luc2/CMV/Neo] and selection under neomycin. HCT116 cells and luciferase-tagged HCT16 cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HT29 cells were cultured in McCoy's 5A Medium supplemented with 10% FBS. SW48 cells were cultured in RPMI-1640 medium supplemented with 1% HEPES, 1% Sodium Pyruvate and 10% FBS. SW620 cells were cultured in L-15 medium supplemented with 10% FBS. All cell lines were mycoplasma-free and were authenticated using short tandem repeat. SW620 cells were cultured in humidified incubators at 37° C. with 100% air. All other cells were cultured in humidified incubators at 37° C. with 5% CO₂.

High glucose DMEM (Cat #10-013-CV), McCoy's 5A medium (Cat #10-050-CV), and RPMI-1640 medium (Cat #10-040-CV) were purchased from Corning Cellgro. L-15 medium was purchased from Thermo Fisher (Cat #11415064). FBS was purchased from Gibco (Cat #10437).

Gene and siRNA Transfection, and Other Treatments

Transfection of pCMV6-A-puro-EGFR, pCMV6-A-puro-EGFR^R451C, pCMV6-A-puro-EGFR^K467Tor pCMV6-A-puro-EGFR^RS492Rwas performed using Lipofectamine 3000. SW620 cells were grown in 6-well plates (3×10⁵cells/well with 2 ml medium) for 24 h and then transfected with a plasmid at 1 μg DNA per well for 48 h. For siRNA transfection, cells were grown in 96-well plates (2×10³HCT116 cells/well or 6×10³HT29 cells/well with 100 μl medium) or 24-well plates (2×10⁴HCT116 cells/well or 6×10⁴HT29 cells/well with 500 μl medium) for 24 h and then transfected with nonspecific scramble siRNA, EGFR siRNA or HER2 siRNA (25 nM) using Lipofectamine RNAiMAX for 48 h. All siRNAs were purchased from Origene. The siRNA sequences Origene catalogue numbers are as follows: scramble siRNA, Cat #SR30004; EGFR siRNA, Cat #SR301357A; and HER2 siRNA, Cat #SR301443A.

MTT Cell Proliferation Assay

Cells were grown in 96-well plates. Each well was seeded with 2×10³HCT116 cells, 6×10³HT29 cells, 5×10³SW48 cells, or 4×10³SW620 cells with 150 l culture medium overnight, treated with solvent, PEPD^G278D(5, 25 or 250 nM), cetuximab (2.75, 27.5 or 275 nM), panitumumab (2.77, 27.7 or 277 nM), or PEPD^G278Dplus cetuximab (250 nM each) in 200 μl medium per well for 24, 48 or 72 h, and then incubated with medium containing 9.2 mM MTT (200 μl/well) at 37° C. for 3 h. The cells were then washed with PBS and mixed with dimethyl sulfoxide (150 μl per well), and cell density was determined by measuring the reduction of MTT to formazan spectroscopically at 570 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek). In experiments involving siRNA transfection, 2×10³HCT116 cells or 6×10³HT29 cells were seeded to each well of 96-well plates overnight and then transfected 25 nM siRNA (scramble siRNA, EGFR siRNA or HER2 siRNA) as described above, followed by MTT assay.

Immunofluorescence Staining and Confocal Microscopy

SW48 cells were grown in chamber slides (4×10⁴cells/well) overnight with or without subsequent treatment with PEPD^G278D(25 nM) and/or chloroquine (25 μM) for up to 6 h. The cells were then washed with ice-cold PBS, fixed with 4% paraformaldehyde for 20 min at room temperature (RT), washed again with ice-cold PBS and blocked with 1% BSA in PBS for 1 h at RT. The cells were then incubated with an EGFR antibody, a His tag antibody for detection of PEPD^G278D, and/or a LAMP1 antibody overnight at 4° C., washed with PBS, incubated with a secondary antibody conjugated to Alexa-Fluor 488 or Alexa-Fluor 647 antibody for 1 h at RT and washed again with PBS. The cells were then examined with a BZ-X700 fluorescence microscope (Keyence) with a S PL FL ELWD ADM 40xC objective. Merged images from Z-stack were organized using the ImageJ software (NIH Image).

PI3K Activity Assay

PI3K activity was measured using a PI3-Kinase Activity ELISA Kit from Echelon Biosciences, following manufacturer's instruction. Briefly, PI3K was pulled down from whole cell lysates using an antibody for PI3K/p85. Each sample was prepared from approximately 1×10⁶cells. The entire immunoprecipitate from each sample was mixed with 30 μl of KBZ reaction buffer, which was then mixed with 30 μl of 10 μM PI(4,5)P2 substrate and incubated for 2 h at 37° C. The kinase reaction was terminated by adding 90 μl of kinase stop solution to each reaction solution, and 60 μl of each mixture was transferred together with 60 μl of PIP3 detector to a well in the incubation plate. After incubation at RT for 1 h, 100 μl per sample from the incubation plate was transferred to the detection plate and incubated for 1 h at RT. The detection plate was washed with TBST, incubated with the HRP-conjugated secondary detector for 30 min, washed again with TBST, and the immobilized HRP was measured by a standard colorimetric assay, using 3,3′,5,5′-tetramethylbenzedine as a substrate and a Synergy 2 Multi-Mode Microplate Reader to record absorbance.

RAS Activity Assay

Ras activity in cell lysates was measured using a Ras Activation ELISA Kit from Cell Biolabs. Briefly, approximately 5×10⁶cells were mixed with 0.5 ml lysis buffer. Cell lysates were cleared by centrifugation at 14,000 g for 10 min at 4° C., and 250 μl per sample was mixed with 10 μl of 0.5 M EDTA with or without 5 μl of either 100× GTPγS or 100× GDP, which was incubated at 37° C. for 30 min with agitation and then mixed with 33 μl of 1 M MgCl₂. The mixture after appropriate dilution was transferred at 100 μl per sample to a well in a 96-well plate immobilized with RAF-1 RAS-binding domain and incubated for 1 h at RT. After washing the wells 5 times with wash buffer, 100 μl of an anti-pan-RAS antibody was added to each well and incubated for 1 h at RT. After another round of wash, 100 μl of a secondary antibody-HRP conjugate was added to each well and incubated for 1 h at RT. After yet another round of wash, 100 μl of substrate solution was added to each well and incubated at RT for 20 min, followed by addition of 100 μl of stop solution to each well. Absorbance at 450 nm in each well was recorded by a Synergy 2 Multi-Mode Microplate Reader.

Measurement of Plasma PEPD^G278DConcentration

Plasma PEPD^G278Dconcentration was measured by ELISA as previously reported [26]. Briefly, 96-well ELISA plates were coated with 100 μl/well of a PEPD antibody (mouse monoclonal, against amino acids 101-305, sc-390042) overnight at 4° C. The plates were then washed three times with PBST and incubated with 200 μl/well of a blocking buffer for at least 2 h at RT. The plates were washed again with PBST and incubated with 100 μl/well of a PEPD standard or a sample, which were appropriately diluted, for 2 h at RT. After another round of wash with PBST, each well was incubated with 100 μl of a detection antibody (an anti-PEPD rabbit polyclonal, ab-86507) for 2 h at RT. After another round of wash with PBST, 100 μl of a secondary antibody-HRP conjugate was added to each well, followed by 1 h incubation at RT. The plates were washed again with PBST three times, and each well was then incubated with 100 μl of a HRP substrate solution. After adequate color development, 100 μl of stop solution was added to each well, and absorbance at 450 nm was recorded by a Synergy 2 Multi-Mode Microplate Reader. Purified recombinant PEPD^G278Dwas used as a standard.

Measurement of AREG and HB-EGF

Concentrations of AREG and HB-EGF in tumor tissues were measured using the Human Amphiregulin/AREG ELISA Kit PicoKine and HB-EGF ELISA Kit, following the manufacturer's instruction. Briefly, 100 μl of standard or tumor tissue homogenate (cleared by centrifugation) was added to a microtiter well pre-coated with anti-human AREG or anti-human HB-EGF and incubated for 2 h at RT. After washing the plate with washing buffer, 100 μl of biotinylated anti-human AREG or biotinylated anti-human HB-EGF were added to each well and incubated for 1.5 h at RT. The microtiter wells were washed 3 times with wash buffer and incubated with 100 μl/well of avidin-biotin-peroxidase complex for 40 min at RT. The microtiter wells were washed 5 times with wash buffer and incubated with 90 l/well color developing reagent for 30 min at RT. After adding 100 μl of stop solution to each well, absorbance at 450 nm was recorded by a Synergy 2 Multi-Mode Microplate Reader.

Preparation of Cell Lysates and Tumor Tissue Homogenates

To prepare whole cell lysates, cells were washed with PBS twice, mixed with 1× cell lysis buffer from Cell Signaling Technology supplemented with 2 mM PMSF and a protease inhibitor cocktail from Roche Applied Science, placed on ice for 10 min, sonicated at 0-4° C. to enhance cell lysis using a Branson Model 450 sonifier, and finally centrifuged at 13,000 g for 10 min at 4° C., and the supernatant fraction is collected as whole cell lysate. Tumor and samples were mixed with RIPA buffer (25 mM Tris-HCl, PH7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS), which was supplemented with 2 mM PMSF, the protease inhibitor cocktail mentioned above, phosphatase inhibitor cocktail 2 and phosphatase inhibitor cocktail 3 from Sigma-Aldrich at 14.3 μl buffer per mg tissue, and homogenized in a Dounce homogenizer. The homogenates were cleared by centrifugation at 13,000 g for 15 min at 4° C.

Western Blotting and Immunoprecipitation (IP)

Protein concentrations in all samples were measured using the BCA Assay Kit. For Western blotting, each sample was mixed with 4× loading dye, heated for 5 min at 95° C., and resolved by SDS-PAGE (8-12.5%). Proteins were transferred to polyvinylidene fluoride membrane, probed with specific antibodies, and detected using either Luminata Classico (Millipore) or Luminata Cresendo (Millipore). For IP, cell lysates (0.5 mg protein/sample) were incubated with a desired antibody overnight at 4° C., followed by incubation of 500 μl sample with 30 μl G-Sepharose beads (2 mg/ml) for 1 h at RT. The beads were washed three times with IP buffer, suspended in 2× SDS loading buffer, boiled for 5 min, and analyzed by Western blotting.

Mouse Study

SCID mice (C.B.17 SCID) were bred by the Laboratory Animal Shared Resource at Roswell Park Comprehensive Cancer Center. Male mice at 7-8 weeks of age were used. All mouse experiments were approved by the Institutional Animal Care and Use Committee at Roswell Park Comprehensive Cancer Center under protocol 1022M. We established subcutaneous tumors by inoculating 1×10⁶HCT116 cells, 4×10⁶HT29 cells, or 2×10⁶SW48 cells to the flank of each mouse in 100 μl of serum free medium. Colon patient-derived xenograft (PDX) 14650 was established from a liver metastatic lesion in a patient treated at Roswell Park Comprehensive Cancer Center. Tumor fragments (˜20 mm³) from donor mice were implanted into the flank of each mouse subcutaneously using a trocar. Orthotopic colon tumors in mice were established by inoculating HCT116 cells stably transfected with firefly luciferase (2×10⁶cells in 50 μl of 50% serum-free medium and 50% Matrigel) to the caecum wall of each mouse. Mice in each tumor model were randomized cage-wise into treatment groups using Research Randomizer (www.randomizer.org). Subcutaneous tumor volume was measured using length ×width²÷2. Tumor volume was measured three times each week. Orthotopic tumor growth was monitored by bioluminescence weekly. Mice were given D-luciferin (150 mg/kg) by ip and anesthetized with isoflurane, and tumor burden was measured by bioluminescence using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). Drug treatment was started when significant tumor growth was detected. EP (0.5 mg/kg) was administered to mice ip once daily. PEPD^G278D(4 mg/kg) was administered to mice ip thrice weekly (Monday, Wednesday, Friday). Aderbasib (60 mg/kg) was administered to mice by gavage once daily. 5-FU (35 mg/kg) was administered to mice ip twice weekly (Monday and Thursday). EP, PEPD^G278Dand 5-FU were administered to mice in PBS. Aderbasib was first dissolved in DMSO and then diluted by PBS (final 5% DMSO by volume). Each agent was administered to mice at 0.1 ml volume per 20 g body weight. When a mouse was given multiple agents on the same day, the agents were dosed at approximately 30 min intervals. The mice were closely monitored for sign of adverse effects and weighed three times each week. Mice were sacrificed 24-48 hours after the last treatment, at which point the tumors were promptly removed, snap frozen and stored at −80° C. for later analysis. Some tumors were fixed in 10% buffered formalin, paraffin embedded, cut at 4 μm, and stained with hematoxylin and eosin (H & E) for histological analysis.

Statistical Analysis

Data were analyzed by two-sided t-test or Mann-Whitney U test for two-group comparison, or one-way analysis of variance (ANOVA) for multi-group comparisons (followed by Tukey multiple comparisons test), using GraphPad Prism 9 software. P value of 0.05 or lower was considered statistically significant. Sample size, mean, SD or SEM, and P value are provided in each figure legend. Each replicate represents an independent sample, not repeated measurement of the same sample.

Example 2

This Example demonstrates that PEPD^G278Dinhibits CRC cells resistant to EGFR MABs.

We compared the response of four human CRC cell lines to cetuximab, panitumumab, and PEPD^G278D, including HCT116, HT29, SW48, and SW620 cells. Both EGFR and HER2 were expressed in HCT116, HT29, and SW48 cells, but their expression levels varied greatly among the cell lines, whereas neither EGFR nor HER2 could be detected in SW620 cells (FIG. 1a). HCT116 cells carry activating mutations of KRAS (G13D) and PIK3CA (H1047R), and HT29 cells carry activating mutations of BRAF (V600E) and PIK3CA (P449T) (FIG. 8). Mutated KRAS, BRAF and PIK3CA are widely believed to drive resistance of CRC cells to EGFR MABs by rendering the cells independent of EGFR.

Both cetuximab and panitumumab inhibited the growth of SW48 cells in a time- and concentration-dependent manner, but neither agent is active in HCT116 cells and HT29 cells (FIG. 1b and FIG. 9a). Cetuximab was also evaluated in SW620 cells and was inactive (FIG. 1b), which is expected, since these cells do not express EGFR. PEPD^G278Dstrongly inhibited the growth of SW48, HCT116, and HT29 cells in a time- and concentration-dependent manner but was ineffective in SW620 cells (FIG. 1b). We previously showed that PEPD^G278Dspecifically targets EGFR and HER2 [19, 20, 22]. Although both PEPD^G278Dand the EGFR MBAs inhibit SW48 cells, the former is more potent than the latter. We next analyzed EGFR, several other RTKs, including HER2, HER3, MET, IGF1R, and several key downstream signaling proteins, including AKT, ERK, and SRC. EGFR and HER2 form heterodimeric signaling units with various RTKs to diversify their oncogenic signaling [28, 29]. The growth-inhibitory activities of the EGFR MABs in SW48 cells were accompanied by decrease in expression and phosphorylation of EGFR as well as decrease in phosphorylation but not expression of MET and ERK (FIG. 1c and FIG. 9b). p-AKT and p-SRC were undetectable in SW48 cells. Decreased phosphorylation of MET and ERK induced by the EGFR MABs in SW48 cells apparently resulted from EGFR inhibition, as neither cetuximab nor panitumumab had any effect on MET and ERK in HCT116 cells and HT29 cells in which EGFR was not inhibited as well as in SW620 cells which do not express EGFR. Cetuximab was also evaluated against HER3 and IGF1R in SW48 cells and showed no effect on the RTKs. Cetuximab showed no effect on any of the signaling proteins in HCT116 and HT29 cells (FIG. 1c). PEPD^G278Dobliterated both expression and tyrosine phosphorylation of EGFR and HER2 in SW48, HCT116 and HT29 cells (FIG. 1c). PEPD^G278Dis more effective than the EGFR MABs in obliterating EGFR in SW48 cells, even though cells were treated by PEPD^G278Dat 25 nM but by the MABs at 275-277 nM. In HCT116, HT29 and SW48 cells, PEPD^G278Dhad no effect on the expression of other signaling proteins but markedly decreased their phosphorylation (FIG. 1c). PEPD^G278Dinhibition of phosphorylation of HER3, IGF1R and MET but not their expression is consistent with its disruption of EGFR association with HER3, MET or IGF1R (FIG. 10). We previously showed that PEPD^G278Dalso disrupts the association of HER2 with each of these RTKs in HER2-positive breast cancer cells [21]. In SW620 cells that lack EGFR and HER2, PEPD^G278Dhad no effect on the phosphorylation or expression of HER3, MET, IGF1R, SRC, AKT, and ERK (FIG. 1c). These results show that by depleting EGFR and HER2 in CRC cells, PEPD^G278Dnot only directly suppresses both RTKs but also indirectly suppresses other RTKs by disrupting their association with EGFR or HER2, thereby causing extensive inhibition of oncogenic signaling. Indeed, siRNA knockdown of EGFR or HER2 also significantly inhibited the growth of HCT116 cells and HT29 cells (FIG. 11a-b). Our results indicate that the inability of EGFR MABs to downregulate EGFR in HCT116 cells and HT29 cells is primarily responsible for their failure to inhibit the growth of these cells, rather than compensatory signaling driven by mutated KRAS, BRAF and PIK3CA. Our results also show that PEPD^G278Dis active in CRC cells overexpressing different levels of EGFR and HER2.

Example 3

PEPD^G278Dabolishes RAS-ERK and PI3K-AKT signaling despite activating mutations in KRAS, BRAF, and PIK3CA

Although HCT116 cells carry activating mutations of KRAS and PIK3CA, and HT29 cells carry activating mutations of BRAF and PIK3CA, and both cell lines are resistant to cetuximab and panitumumab, as described above, the inhibitory activities of PEPD^G278Din HCT116 cells and HT29 cells were similar to that in SW48 cells whose KRAS, BRAF and PIK3CA are not mutated. ERK and AKT are downstream of KRAS and PIK3CA, respectively. PEPD^G278Dcaused marked loss of phosphorylation of ERK and AKT in both HCT116 and HT29 cells (FIG. 1c). MEK is upstream of ERK, and PEPD^G278Dalso markedly decreased MEK phosphorylation in both HCT116 and HT29 cells (FIG. 2a). The loss of phosphorylation of MEK, ERK and AKT apparently resulted from PEPD^G278Dtargeting EGFR and HER2, as PEPD^G278Dwas inactive in SW620 cells and cetuximab was active only in SW48 cells (FIGS. 1c and 2a).

It was previously shown that oncogenic RAS mutants regulate basal effector pathway signaling, while WT RAS in the same cells mediates signaling downstream of activated RTKs [30]. It was also shown that the gain of function of PIK3CA mutants is enabled by activated RAS or PI3K/p85-mediated binding to activated RTKs [31, 32]. Notably, only one allele of each of the KRAS and PIK3CA genes in HCT116 cells is mutated, and only one allele of each of the BRAF and PIK3CA genes in HT29 cells is mutated (FIG. 8). Moreover, HRAS and NRAS are also expressed in both HCT116 and HT29 cells as well as in SW48 and SW620 cells (FIG. 2a). These results provide an explanation for why PEPD^G278Dis able to strongly inactivate MEK, ERK and AKT in HCT116 cells and HT29 cells. We also found that PEPD^G278Dstrongly downregulates the expression of both HRAS and NRAS in HCT116, HT29 and SW48 cells but not in SW620 cells, whereas cetuximab had no effect on the expression of HRAS and NRAS in any of the cell lines (FIG. 2a). Since cetuximab only targets EGFR, the above results suggested that downregulation of HRAS and NRAS by PEPD^G278Dmight result from HER2 depletion. Indeed, siRNA silence of HER2 but not EGFR resulted in loss of HRAS and NRAS (FIG. 11a). However, neither PEPD^G278Dnor cetuximab regulated the expression of WT or mutated KRAS (FIG. 2a). Consistent with the changes in the signaling molecules described above, total RAS and PI3K activities were strongly inhibited by PEPD^G278Din HCT116 and HT29 cells as well as in SW48 cells but not in SW620 cells (FIG. 2b-c). There was little difference among HCT116, HT29 and SW48 cells with regard to percentage of inhibition of RAS and PI3K by PEPD^G278D, as RAS activity was inhibited by 73% in HCT116 cells and 75% in both HT29 and SW48 cells, and PI3K activity was inhibited by 74% in HCT116 cells, 71% in HT29 cells, and 76% in SW48 cells. Notably, basal RAS activity is much higher in HCT116 and SW620 cells than in HT29 and SW48 cells, consistent with HCT116 cells carrying KRAS^G12D(FIG. 8) and SW620 cells carrying KRAS^Gl2V[27], and basal PI3K activity is much higher in HCT116 and HT29 cells than in SW48 and SW620, consistent with HCT116 cells carrying PIK3CA^H1047Rand HT29 cells carrying PIK3CA^P449T(FIG. 8). Also, the remaining RAS and PI3K activities after PEPD^G278Dtreatment were still higher in HCT116 and HT29 cells than in SW48 cells. Collectively, our results show that PEPD^G278Dstrongly inhibits the RAS-MEK-ERK and PI3K-AKT signaling pathways by depleting EGFR and HER2, even if CRC cells harbor activating mutations of KRAS, BRAF and/or PIK3CA. Our results also indicate that PEPD^G278Daccomplishes this feat by abolishing both canonical function (tyrosine kinase) and non-canonical function (scaffolding—heterodimerization with other RTKs) of EGFR and HER2 as well as abolishing HER2 regulation of HRAS and NRAS.

Example 4

PEPD^G278DAlso Targets EGFR Mutants that Occur in CRC Patients

While EGFR is not mutated in SW48, HCT116 and HT29 cells [27], several acquired mutations in the extracellular domain of EGFR have been reported in CRC patients following cetuximab treatment, including R451C, K467T, and S492R, each of which prevents cetuximab binding and confers resistance to cetuximab [33, 34]. EGFR^R451Cand EGFR^K467Talso bind poorly to panitumumab [33]. However, these mutations locate far from the site (amino acids #166-310) to which PEPD^G278Dbinds [20]. Because SW620 cells do not express EGFR, we transfected each EGFR mutant as well as WT EGFR into these cells and then treated the cells with solvent or PEPD^G278D(25 nM for 48 h). Each EGFR mutant was strongly downregulated by PEPD^G278D, showing loss of both expression and phosphorylation, and the extent of downregulation of each mutant by PEPD^G278Dis very similar to that of WT EGFR (FIG. 2d). Thus, mutations in EGFR which occur in CRC patients do not interfere with PEPD^G278Dtargeting of EGFR.

Example 5
EGFR Ligands Slow PEPD^G278DInduction of EGFR Internalization and Lysosomal Degradation

PEPD^G278Dcauses depletion of both EGFR and HER2, but HER2 depletion was much faster than that of EGFR in cells cultured in medium with 10% serum. In HCT116, HT29, and SW48 cells, HER2 level decreased markedly after 3 h of PEPD^G278Dtreatment, whereas EGFR level showed no decrease even after 6 h of PEPD^G278Dtreatment, although it showed profound decrease at 24 h (FIG. 3a). However, if the cells were cultured in serum-free medium, both EGFR and HER2 showed marked decrease after 3 h treatment with PEPD^G278D(FIG. 3a). We previously showed that epidermal growth factor (EGF), a high affinity EGFR ligand, competes with PEPD for binding to EGFR [26]. Notably, EGFR ligands bind to subdomains 1 and 3 in EGFR extracellular domain [35, 36], whereas PEPD^G278Dbinds to subdomain 2 in EGFR [20]. Adding HB-EGF, another high affinity EGFR ligand, to culture medium without serum mimicked the effect of serum on EGFR depletion induced by PEPD^G278D, while HB-EGF itself did not modulate the expression of EGFR or HER2 (FIG. 3a). No HER2 ligand, other than PEPD or PEPD^G278D, is known. These results suggest that EGFR ligands from serum interfere with PEPD^G278Dtargeting of EGFR. We also found that cetuximab attenuates the growth-inhibitory activity of PEPD^G278Din both HCT116 cells and HT29 cells (FIG. 12a), which likely resulted from cetuximab competing with PEPD^G278Dfor EGFR binding. Notably, cetuximab binds to extracellular subdomain 3 of EGFR [37].

We previously showed that PEPD^G278Dinduces HER2 internalization and lysosomal degradation [21]. Here, we show that PEPD^G278Dalso induces EGFR internalization and lysosomal degradation. We focused on SW48 cells, taking advantage of their high EGFR level. SW48 cells were cultured in serum-free medium. PEPD^G278Dbinding to EGFR and subsequent EGFR trafficking were analyzed by immunofluorescence staining and confocal microscopy. PEPD^G278Dbound abundantly to cell membrane and colocalized with EGFR after 15 min of treatment, but at 6 h, neither PEPD^G278Dnor EGFR remained on cell membrane, with residual amount of PEPD^G278Dbut no EGFR detected intracellularly (FIG. 3b). Next, cells were treated with PEPD^G278Dand/or chloroquine, the latter of which is a lysosome inhibitor. In the absence of chloroquine, PEPD^G278Dinduced EGFR internalization, and the internalized EGFR colocalized with LAMP1I, a lysosome marker, but at 6 h of treatment, almost no EGFR could be detected (FIG. 3c). However, chloroquine blocked EGFR degradation induced by PEPD^G278D(FIG. 3c). Collectively, our results show that PEPD^G278Dinduces EGFR internalization and degradation in the lysosome but EGFR ligands slow this process by interfering with PEPD^G278Dbinding to EGFR.

Example 6

PEPD^G278DFails to Inhibit Tumors that Overexpress a High-Affinity EGFR Ligand

We next compared the antitumor activities of PEPD^G278Dand cetuximab in vivo. PEPD^G278Dis degraded in vivo by coagulation proteases, but EP, a clinically used anticoagulant, inhibits PEPD^G278Ddegradation [38]. EP itself has no antitumor activity but combining EP with PEPD^G278Dallows therapeutically relevant plasma concentrations of PEPD^G278Dto be achieved for inhibition of tumors overexpressing EGFR and/or HER2 [20, 22]. We inoculated human CRC cells to immunocompromised mice subcutaneously, and the tumor-bearing mice were randomized for treatment with EP, EP plus PEPD^G278D, or cetuximab. Based on previous studies, EP was administered to the mice at 0.5 mg/kg daily by intraperitoneal injection (ip); PEPD^G278Dwas administered at 4 mg/kg ip three times weekly; and cetuximab was administered at 15 mg/kg ip twice weekly. Both PEPD^G278Dand cetuximab strongly inhibited the growth of SW48 tumors and at the end of treatment inhibiting tumor growth by 91.5% and 85.8%, reactively, but neither agent inhibited the growth of HCT116 tumors and HT29 tumors (FIG. 4a). Escalating PEPD^G278Dto 8 mg/kg did not inhibit tumor growth either (FIG. 12b). Tumors were collected 24 h after the final dose of each agent and select signaling proteins were analyzed. In SW48 tumors, PEPD^G278Ddecreased the expression and phosphorylation of both EGFR and HER2, while cetuximab only decreased the expression and phosphorylation of EGFR, and both agents also decreased ERK phosphorylation (FIG. 4b). Similar results were shown in cultured SW48 cells as described before. However, neither PEPD^G278Dnor cetuximab had any effect on EGFR, HER2 and ERK in HCT116 and HT29 tumors (FIG. 4b). Yet, in cultured HCT116 and HT29 cells as described before, while cetuximab was inactive, PEPD^G278Dstrongly reduced the expression and phosphorylation of both EGFR and HER2 and decreased ERK phosphorylation.

Lack of inhibitory activity of PEPD^G278Din HCT116 tumors and HT29 tumors was not due to lack of PEPD^G278Ddelivery, as plasma concentrations of PEPD^G278Dwere high and similar in mice bearing SW48 tumors and HCT116 tumors (FIG. 4c). We analyzed all seven known EGFR ligands (soluble form) in the tumor tissues but detected only AREG and HB-EGF. AREG is a low affinity EGFR ligand, and its affinity for EGFR is approximately 50 fold lower than that of HB-EGF [39]. AREG level was high in all three types of tumors (FIG. 4d). HB-EGF level was 13.6-14.6 fold higher in HT29 and HCT116 tumors than in SW48 tumors (FIG. 4e). This suggests that excessive tumor-generated HB-EGF might prevent PEPD^G278Dfrom binding to EGFR, and also suggests that EGFR signaling remains important to the tumors carrying activating mutations of KRAS, BRAF and/or PIK3CA. PEPD^G278Dalso failed to downregulate HER2 in tumors expressing high level of HB-EGF. HB-EGF does not bind to HER2, and we showed previously that PEPD^G278Ddisrupts the HER2-EGFR heterodimer even when EGF is bound to EGFR [22]. However, it is possible that without inhibiting EGFR, the impact of PEPD^G278Don HER2 may be negated by rapid tumor growth.

Example 7
Aderbasib Restores the Antitumor Activity of PEPD^G278Din Tumors Overexpressing HB-EGF

Aderbasib inhibits the shedding of all EGFR ligands by inhibiting ADAM10 and ADAM17 as mentioned before. We evaluated the antitumor activity of aderbasib as a single agent or in combination with EP plus PEPD^G278Din HCT116 and HT29 tumors. Tumor-bearing mice were treated with EP, aderbasib, or the combination of aderbasib with EP and PEPD^G278D. As in previous experiments, EP was administered at 0.5 mg/kg ip daily, and PEPD^G278Dwas administered at 4 mg/kg ip three times weekly. Aderbasib was administered at 60 mg/kg by gavage daily. In HCT116 and HT29 tumors, aderbasib alone was ineffective, but combining aderbasib with EP plus PEPD^G278Dinhibited tumor growth by 63.3% and 54.4% respectively at the end of treatment (FIG. 5a). The combination treatment was less effective when aderbasib was reduced to 30 mg/kg and became ineffective when it was reduced to 15 mg/kg (FIG. 13a-c). Mice treated with aderbasib alone or the combination regimen did not show signs of toxicity.

Aderbasib caused marked decrease in soluble HB-EGF level in the tumor tissues (FIG. 5b). In both tumor models, neither EP nor aderbasib had any effect on the expression or phosphorylation of the proteins analyzed, but combining aderbasib with EP and PEPD^G278Dcaused profound loss of both expression and phosphorylation of EGFR and HER2, loss of phosphorylation but not expression of all other RTKs and downstream signaling proteins analyzed, including HER3, IGF1R, MET, SRC, AKT, ERK, and MEK, loss of expression of NRAS and HRAS, and activation of caspase 3 (FIG. 5c). These results show that by blocking shedding of HB-EGF from tumor cells, aderbasib enables PEPD^G278Dto engage its targets and to exert its antitumor activity. Although not measured, aderbasib probably also blocked the shedding of AREG from the tumor cells.

Notably, in tumors treated by the triple combination (EP, aderbasib and PEPD^G278D), low levels of p-AKT, p-ERK and p-MEK remained despite profound loss of both expression and phosphorylation of EGFR and HER2 induced by PEPD^G27D, suggesting that mutated PIK3CA, KRAS and BRAF may sustain a low level of signaling despite depletion of EGFR and HER2. Likewise, in cultured HCT116 and HT29 cells, despite profound loss of both EGFR and HER2 and marked inhibition of both RAS and PI3K activities by PEPD^G278D, residual RAS and PI3K activities remain as mentioned before.

Example 8
Adding 5-FU to the PEPD^G278D-Based Combination Treatment Enhances Therapeutic Outcome

Because 5-FU, an antimetabolite, is commonly used in CRC treatment, we asked whether combining 5-FU with the PEPD^G278D-based combination regime described above enhances treatment outcome. Thus, tumor-bearing mice were treated with EP, or the combination of EP, 5-FU, aderbasib and PEPD^G278D. As in other experiments, EP was administered at 0.5 mg/kg ip daily, PEPD^G278Dwas administered at 4 mg/kg ip three times weekly, and aderbasib was administered by gavage at 60 mg/kg daily. 5-FU was administered to mice at 35 mg/kg ip once every 3-4 days, which was not toxic in a dose-finding experiment. We first evaluated the combination regimen in mice bearing subcutaneous HCT 116 and HT29 tumors. The combination treatment was highly effective against both types of tumors, inhibiting tumor growth by 72.4% (HCT 116 tumors) and 69.4% (HT29 tumors) at the end of treatment (FIG. 6a-b), which is more efficacious than the combination minus 5-FU as described before. 5-FU as a single agent or 5-FU in combination with EP and PEPD^G278Dwithout aderbasib was ineffective (FIG. 14). The combination regimen was also evaluated in a CRC PDX (PDX14650) which harbors KRAS^G12D(homozygous) and generates high level of HB-EGF (FIG. 15). The combination regime markedly decreased tumor soluble HB-EGF level (FIG. 15) and inhibited tumor growth by 83.2% at the end of treatment (FIG. 6c). Adverse effects were not detected in the mice in any of the tumor models. Tumors in the different models were collected 24 or 48 h after final treatment. In all the tumor models, the combination treatment caused profound loss in both expression and phosphorylation of EGFR and HER2, loss of phosphorylation but not expression of all other RTKs and downstream signaling proteins measured, including HER3, IGF1R, MET, SRC, AKT, MEK, ERK, loss of expression of HRAS and NRAS, and activation of caspase 3 (FIG. 6d). These results are similar to that obtained from tumors treated by aderbasib plus EP and PEPD^G278Dand from cultured cells treated by PEPD^G278Das a single agent, as described before. Thus, 5-FU enhances tumor inhibition when combined with the other agents but does not interfere with the inhibition of oncogenic signaling by PEPD^G278Dand does not interfere with aderbasib inhibition of EGFR ligand shedding by tumor cells. 5-FU is known to exert its antitumor activity by causing misincorporation of fluoronucleotides into RNA and DNA and inhibiting the nucleotide synthetic enzyme thymidylate synthase.

We also evaluated the combination regimen in mice bearing orthotopic HCT116 tumors. HCT116 cells stably expressing firefly luciferase were inoculated to the cecum of mice. Tumor growth was monitored by bioluminescence imaging (FIG. 16a). Mice were randomized to EP or the combination regimen. Treatments with EP, aderbasib, PEPD^G278Dand 5-FU were the same as described before and were started on 22, 26, 27 and 28 days after cell inoculation, respectively. Tumor burden was not significantly different between the control and combination treatment group at the beginning of treatment (day 25), but tumor burden became significantly and consistently lower in the combination treatment group (FIG. 7a). The mean tumor bioluminescence intensity in the combination treatment group was consistently nearly 2 orders of magnitude lower than that in the control. The experiment was terminated on day 57, when several mice in the control group became moribund. Necropsy showed primary tumors in the cecum, local metastasis (peritoneal tumors), and liver metastasis. However, macroscopic tumors were found only in mice showing bioluminescence signals of >3.2×10⁸photons per second. Six of the 11 mice in the control (54.6%) and 2 of the 12 mice in the combination treatment (16.7%) showed cecum and/or peritoneal tumors (FIG. 7b). Average tumor weight (cecum and peritoneal tumors) in the combination treatment was only 8.8% of that in the control (FIG. 7c). Four of the 11 mice (36.4%) in the control showed liver metastasis, but only 1 of the 12 mice (8.3%) in the combination treatment showed liver metastasis (FIG. 7d). Representative primary and metastatic tumors are shown in FIG. 16b. All tumors were verified by histological analysis, and representative images are shown in FIG. 16c. Adverse effects of the treatments were not detected. As in other experimental models described before, the combination treatment caused profound loss in both expression and phosphorylation of EGFR and HER2, loss of phosphorylation but not expression of HER3, IGF1R, MET, SRC, AKT, MEK and ERK, loss of expression of HRAS and NRAS, and activation of caspase 3 (FIG. 7e).

Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.

The following reference listing is not an indication that any particular reference is material to patentability:

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DEPLETING EGFR AND HER2 OVERCOMES RESISTANCE TO EGFR INHIBITORS IN COLORECTAL CANCER

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