Patent Application
20240307414
This document includes a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-02014WO1_ST25.txt. The ASCII text file, created on Nov. 5, 2021, is 7 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
This document relates to methods and materials for assessing and/or treating mammals (e.g., humans) having prostate cancer. For example, the methods and materials provided herein can be used to determine whether or not a prostate cancer is likely to respond to a particular cancer treatment (e.g., treatment with one or more anti-androgen agents such as inhibitors of androgen biosynthesis and androgen receptor antagonists). Also provided are methods and materials for using one or more cancer treatments to treat a mammal (e.g., a human) identified as likely to respond to a particular cancer treatment.
Prostate cancer (PC) is the second most frequently diagnosed and the second most deadly cancer type for men in the United States (Siegel et al., CA Cancer J. Clin., 68, 7-30 (2018)). Approximately 10-20% of patients will develop metastatic prostate cancer (mPC) associated with a reduced survival rate. Androgen deprivation therapies (ADTs), including anti-androgen agents such as androgen biosynthesis inhibitors and antagonists for the androgen receptor (AR), the primary transcriptional regulator in normal and cancerous prostate cells, are the first-line systemic treatment of mPC (Knudsen et al., Clin. Cancer Res., 15, 4792-4798 (2009)). Despite initial response to ADT, a majority of these patients eventually develop hormone-refractory PC, castration-resistant prostate cancer (CRPC; Knudsen et al., Clin. Cancer Res., 15, 4792-4798 (2009); and Spratt et al., Prostate, 75, 175-182 (2015)). Second-generation ADT drugs such as abiraterone acetate (Abi), a cytochrome P450 17A1 (CYP17A1) inhibitor, have been shown to extend overall survival significantly (Azad et al., Clin. Cancer Res., 21, 2315-2324 (2015); and Romanel et al., Sci. Transl. Med., 7, 312re310 (2015)). However, at least 30% of patients do not respond to initial Abi treatment and nearly all patients will eventually develop acquired resistance.
This document provides methods and materials related to assessing and/or treating prostate cancer. In some cases, this document provides methods and materials for determining whether or not a mammal (e.g., a human) having prostate cancer is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent), and, optionally, administering to the mammal one or more cancer treatments selected based, at least in part, on whether or not the mammal is likely to respond to a particular cancer treatment. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer can be assessed to determine if the mammal is likely to respond to a particular cancer treatment based, at least in part, on the presence or absence of an increased level of expression of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more) polypeptides in the sample.
As described herein, prostate cancers that respond to abiraterone (Abi responders) exhibit differential gene expression as compared to prostate cancers that do not respond to abiraterone (Abi non-responders). For example, increased expression of a cyclin A2 (CCNA2) nucleic acid (e.g., resulting in increased level of expression of CCNA2 polypeptides), increased expression of a cyclin B1 (CCNB1) nucleic acid (e.g., resulting in increased level of expression of CCNB1 polypeptides), increased expression of a cyclin B2 (CCNB2) nucleic acid (e.g., resulting in increased level of expression of CCNB2 polypeptides), increased expression of a protein regulator of cytokinesis 1 (PRC1) nucleic acid (e.g., resulting in increased level of expression of PRC1 polypeptides), increased expression of a structural maintenance of chromosomes protein 2 (SMC2) nucleic acid (e.g., resulting in increased level of expression of SMC2 polypeptides), increased expression of a discs large-associated protein 5 (DLGAP5) nucleic acid (e.g., resulting in increased level of expression of DLGAP5 polypeptides), increased expression of a epithelial cell transforming 2 (ECT2) nucleic acid (e.g., resulting in increased level of expression of ECT2 polypeptides), increased expression of a F-box only protein 5 (FBXO5) nucleic acid (e.g., resulting in increased level of expression of FBXO5 polypeptides), increased expression of a cyclin dependent kinase 1 (CDK1) nucleic acid (e.g., resulting in increased level of expression of CDK 1 polypeptides), increased expression of a non-SMC condensin I complex subunit G (NCAPG) nucleic acid (e.g., resulting in increased level of expression of NCAPG polypeptides), and increased expression of a kinesin family member 4A (KIF4A) nucleic acid (e.g., resulting in increased level of expression of KIF4A polypeptides) can be used to identify prostate cancer patients as having abiraterone resistance. Also as described herein, one or more DNA topoisomerase 2-alpha (TOP2A) inhibitors, one or more cyclin-dependent kinase (CDK) 4/6 inhibitors, one or more Mitogen-Activated Protein Kinase Kinase (MEK) inhibitors, and/or one or more pan-CDK inhibitors can sensitize prostate cancers to one or more anti-androgen agents. These results demonstrate that the presence or absence of an increased level of expression of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more) polypeptides (e.g., an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides) in a sample from a mammal having prostate cancer can be used to determine anti-androgen agent (e.g., abiraterone) responsiveness of that mammal.
Having the ability to identify a mammal having prostate cancer as being likely to respond to a particular cancer treatment based, at least in part, on the presence or absence of an increased level of expression of one or more polypeptides provides a unique and unrealized opportunity to provide an individualized approach in selecting effective prostate cancer therapies.
In general, one aspect of this document features methods for assessing a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample from a mammal having prostate cancer, a presence or absence of an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; (b) classifying the mammal as being unlikely to respond to an anti-androgen agent if the presence of the increased level is detected; and (c) classifying the mammal as being likely to respond to the anti-androgen agent if the absence of the increased level is detected. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The method can include detecting the presence of the increased level of the polypeptide. The method can include classifying the mammal as being unlikely to respond to the anti-androgen agent. The method can include detecting the absence of the increased level of the polypeptide. The method can include classifying the mammal as being likely to respond to the anti-androgen agent. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide. The prostate cancer can be a metastatic prostate cancer. The method can detect the presence or absence of an increased level of expression of three of the polypeptides. The method can detect the presence or absence of an increased level of expression of five of the polypeptides. The method can detect the presence or absence of an increased level of expression of seven of the polypeptides. The method can detect the presence or absence of an increased level of expression of nine of the polypeptides. The method can detect the presence or absence of an increased level of expression of eleven of the polypeptides. The detecting can include a clustering analysis. The clustering analysis can be a machine learning based clustering analysis.
In another aspect, this document features methods for treating a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample obtained from a mammal having prostate cancer, an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; and (b) administering a cancer treatment to the mammal, where the cancer treatment is not an anti-androgen agent. The method can include detecting the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The cancer treatment can include a radiation treatment. The cancer treatment can include administering to the mammal a cancer drug that is not an anti-androgen agent. The cancer drug that is not an anti-androgen agent can be docetaxel, cabazitaxel, mitoxantrone, estramustine, doxorubicin, palbociclib, ribociclib, abemaciclib, PD-0325901, PHA-793887, or any combinations thereof.
In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering a cancer treatment to a mammal having prostate cancer and identified as having an increased level of expression of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, or a KIF4A polypeptide in a sample obtained from the mammal, where the cancer treatment is not an anti-androgen agent. The mammal can be identified as having an increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The cancer treatment can include a radiation treatment. The cancer treatment can include administering to the mammal a cancer drug that is not an anti-androgen agent. The cancer drug that is not an anti-androgen agent can be docetaxel, cabazitaxel, mitoxantrone, estramustine, doxorubicin, palbociclib, ribociclib, abemaciclib, PD-0325901, PHA-793887, or any combinations thereof.
In another aspect, this document features methods for treating a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample obtained from a mammal having prostate cancer, an absence of an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; and (b) administering an anti-androgen agent to the mammal. The method can include detecting the absence of the level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering an anti-androgen agent to a mammal having prostate cancer and identified as lacking an increased level of expression of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, a KIF4A polypeptide, or any combinations thereof in a sample obtained from the mammal. The mammal can be identified as lacking the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample obtained from a mammal having prostate cancer, a presence of an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; (b) administering a TOP2A inhibitor to the mammal to increase the sensitivity of prostate cancer cells within the mammal to an anti-androgen agent; and (c) administering the anti-androgen agent to the mammal. The method can include detecting the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The TOP2A inhibitor can be mitoxantrone, doxorubicin, teniposide, daunorubicin, amsacrine, ellipticines, aurintricarboxylic acid, or HU-331. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering a TOP2A inhibitor and an anti-androgen agent to a mammal having prostate cancer and identified as having an increased level of expression of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, a KIF4A polypeptide, or a combination thereof in a sample obtained from the mammal. The mammal can be identified as having the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The TOP2A inhibitor can be mitoxantrone, doxorubicin, teniposide, daunorubicin, amsacrine, ellipticines, aurintricarboxylic acid, or HU-331. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample obtained from the mammal, a presence of an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; (b) administering a cyclin-dependent kinase (CDK) 4/6 inhibitor to the mammal to increase the sensitivity of prostate cancer cells within the mammal to an anti-androgen agent; and (c) administering the anti-androgen agent to the mammal. The method can include detecting the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The CDK 4/6 inhibitor can be palbociclib, abemaciclib, or ribociclib. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering a CDK 4/6 inhibitor and an anti-androgen agent to a mammal having prostate cancer and identified as having an increased level of expression of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, a KIF4A polypeptide, or a combination thereof in a sample obtained from the mammal. The mammal can be identified as having the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The CDK 4/6 inhibitor can be palbociclib, abemaciclib, or ribociclib. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a mammal having prostate cancer. The methods can include, or consist essentially of, (a) detecting, in a sample obtained from a mammal having prostate cancer, a presence of an increased level of expression of a polypeptide selected from the group consisting of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, and a KIF4A polypeptide, or a combination thereof; (b) administering a pan-CDK inhibitor to the mammal to increase the sensitivity of prostate cancer cells within the mammal to an anti-androgen agent; and (c) administering the anti-androgen agent to the mammal. The method can include detecting the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The pan-CDK 4/6 inhibitor can be PHA-793887. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering a pan-CDK inhibitor and an anti-androgen agent to a mammal having prostate cancer and identified as having an increased level of expression of a CCNA2 polypeptide, a CCNB1 polypeptide, a CCNB2 polypeptide, a PRC1 polypeptide, a SMC2 polypeptide, a DLGAP5 polypeptide, an ECT2 polypeptide, a FBXO5 polypeptide, a CDK1 polypeptide, a NCAPG polypeptide, a KIF4A polypeptide, or a combination thereof in a sample obtained from the mammal. The mammal can be identified as having the increased level of expression of the CCNA2 polypeptide, the CCNB1 polypeptide, the CCNB2 polypeptide, the PRC1 polypeptide, the SMC2 polypeptide, the DLGAP5 polypeptide, the ECT2 polypeptide, the FBXO5 polypeptide, the CDK1 polypeptide, the NCAPG polypeptide, and the KIF4A polypeptide. The mammal can be a human. The sample can include cancer cells of the prostate cancer. The pan-CDK 4/6 inhibitor can be PHA-793887. The anti-androgen agent can be leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, or darolutamide.
Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials involved in assessing and/or treating mammals (e.g., humans) having prostate cancer. In some cases, the methods and materials provided herein can be used to determine whether or not a mammal having prostate cancer is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent). For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer can be assessed for the presence or absence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides to determine whether or not the mammal is likely to respond to an anti-androgen agent (e.g., abiraterone). In some cases, the methods and materials provided herein also can include administering one or more cancer treatments to a mammal having prostate cancer to treat the mammal (e.g., one or more cancer treatments selected based, at least in part, on whether or not the mammal is likely to respond to a particular cancer treatment such as an anti-androgen agent).
A mammal (e.g., a human) having prostate cancer can be assessed to determine whether or not the cancer is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent) by detecting the presence or absence of an increased level of expression of one or more polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. As described herein, the presence of an increased level of expression of one or more polypeptides in a sample obtained from the mammal can be used to determine whether or not that mammal is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent). For example, the presence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK 1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample obtained from a mammal having prostate cancer can be used to identify that mammal as being unlikely to respond to one or more anti-androgen agents. Also as demonstrated herein, one or more TOP2A inhibitors and/or one or more CDK 4/6 inhibitors can be used to sensitize prostate cancers to one or more anti-androgen agents. For example, one or more TOP2A inhibitors, one or more CDK 4/6 inhibitors, one or more MEK inhibitors, and/or one or more pan-CDK inhibitors can be administered to a mammal having prostate cancer and identified as having the presence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides to sensitize the mammal to one or more anti-androgen agents, and optionally, the mammal can be administered one or more anti-androgen agents to treat the mammal.
Any appropriate mammal having prostate cancer can be assessed and/or treated as described herein. In some cases, a mammal having prostate cancer can have undergone no prior treatment for the prostate cancer. In some cases, a mammal having prostate cancer can have undergone treatment for the prostate cancer. For example, a mammal have prostate cancer can have undergone a surgical treatment for the prostate cancer. For example, a mammal having prostate cancer can have been administered one or more anti-cancer agents (e.g., one or more anti-androgen agents such as abiraterone and/or one or more cancer drugs that are not an anti-androgen agent such as docetaxel). Examples of mammals that can have prostate cancer and can be assessed and/or treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, rats, and Guinea pigs, hamsters. In some cases, a mammal can be a male mammal. For example, a male human having prostate cancer can be assessed and/or treated as described herein.
When assessing and/or treating a mammal (e.g., a human) having prostate cancer as described herein, the prostate cancer can be any type of prostate cancer. A prostate cancer can be any stage of prostate cancer (e.g., stage I, stage II, stage III, or stage IV). A prostate cancer can be any grade of prostate cancer (e.g., grade 1, grade 2, or grade 3). A prostate cancer can have any Gleason score. In some cases, a prostate cancer can be a primary cancer (e.g., a localized primary cancer). In some cases, a prostate cancer can have metastasized. In some cases, a prostate cancer can be castration-sensitive prostate cancer (CSPC). In some cases, a prostate cancer can be castration-resistant prostate cancer (CRPC). In some cases, a prostate cancer can be hormone-refractory prostate cancer (HRPC).
In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having prostate cancer. Any appropriate method can be used to identify a mammal as having prostate cancer. For example, physical examination (e.g., a digital rectal examination (DRE)), laboratory testing (e.g., blood tests for prostate-specific antigen (PSA) test), imaging techniques (e.g., ultrasound, magnetic resonance imaging (MRI), bone scan, computerized tomography (CT) scan, and positron emission tomography (PET) scan), and biopsy techniques can be used to identify a mammal (e.g., a human) as having prostate cancer.
In some cases, a mammal (e.g., a human) having prostate cancer can be assessed to determine whether or not the cancer is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent such as abiraterone) based, at least in part, on the presence or absence of an increased level of expression of one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, or more) polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. The term “increased level” as used herein with respect to a level of a polypeptide refers to any level that is greater than a reference level of that polypeptide. The term “reference level” as used herein with respect to a polypeptide refers to the level of that polypeptide typically observed in a sample (e.g., a control sample) from one or more comparable mammals (e.g., humans of comparable age) that do not have prostate cancer. In some cases, a reference level can be obtained using a machine learning based clustering method. Control samples can include, without limitation, comparable samples from mammals that do not have prostate cancer. Examples of polypeptides that can have increased levels of expression in a sample from a mammal having prostate cancer include, without limitation, CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, KIF4A polypeptides, androgen receptor (AR) polypeptides (e.g., AR splice variant polypeptides), PSA polypeptides, TOP2A polypeptides, ERV fusion polypeptides, tumor protein P53 (TP53) polypeptides, speckle type BTB/POZ Protein (SPOP) polypeptides, and forkhead box A1 (FOXA1) polypeptides. In some cases, an increased level of expression of a polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater relative to a reference level of that polypeptide. In some cases, when control samples have an undetectable level of a polypeptide, an increased level can be any detectable level of that polypeptide. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is an increased level. In some cases, a polypeptide having an increased level of expression in a sample from a mammal having prostate cancer can be as described in Example 1.
In some cases, the methods described herein can include detecting the presence or absence of an increased level of expression of a panel of polypeptides. For example, a panel of polypeptides can include any two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or more) of the polypeptides described herein. In some cases, the presence or absence of two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, or more) polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human) having prostate cancer can be used to determine whether or not that mammal is likely to respond to a particular cancer treatment (e.g., an anti-androgen agent).
Any appropriate method can be used to detect the presence or absence of an increased level of expression of one or more polypeptides within a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human). In some cases, a level of polypeptide expression within a sample can be determined by detecting the presence, absence, or level of the polypeptide in the sample. For example, immunoassays (e.g., immunohistochemistry (IHC) techniques, western blotting techniques, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation, and immunofluorescence such as immunofluorescence coupled flow cytometry), mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), enzyme-linked immunosorbent assays (ELISAs), and radio-immunoassays can be used to determine the presence, absence, or level of a polypeptide in a sample. In some cases, a level of polypeptide expression within a sample can be determined by detecting the presence, absence, or level of mRNA encoding the polypeptide in the sample. For example, polymerase chain reaction (PCR)-based techniques such as quantitative RT-PCR techniques, nanoString ncounter techniques, gene expression panels or arrays (e.g., next generation sequencing (NGS) such as RNA-seq, miRNAseq, amplicon sequencing, and nanopore sequencing), in situ hybridization (ISH) such as fluorescence in situ hybridization (FISH), and gel electrophoresis can be used to determine the presence, absence, or level of mRNA encoding the polypeptide in the sample. In some cases, a level of polypeptide expression within a sample can be determined using a machine learning based clustering algorithm. Examples of machine learning based clustering algorithms include, without limitation, kmeans, hierarchical clustering, support vector machines, decision trees, random forests, mean-shift clustering, density-based spatial clustering of applications with noise, and expectation-maximization (EM) clustering using Gaussian mixture models (GMM). In some cases, the presence or absence of an increased level of expression of one or more polypeptides within a sample from a mammal having prostate cancer can be determined as described in Example 1.
In some cases, a mammal (e.g., a human) having prostate cancer can be identified as being unlikely to respond to a particular cancer treatment (e.g., an anti-androgen agent such as abiraterone) based, at least in part, on the presence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK 1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. For example, a mammal having prostate cancer can be identified as being unlikely to respond to one or more anti-androgen agents (e.g., abiraterone) based, at least in part, on the presence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK 1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample obtained from the mammal.
In some cases, a mammal (e.g., a human) having prostate cancer can be identified as being likely to respond to a particular cancer treatment (e.g., an anti-androgen agent such as abiraterone) based, at least in part, on the absence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK 1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. For example, a mammal having prostate cancer can be identified as being likely to respond to one or more anti-androgen agents (e.g., abiraterone) based, at least in part, on the absence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample obtained from the mammal.
In some cases, a mammal (e.g., a human) having prostate cancer can be identified as being likely to respond to a particular cancer treatment (e.g., a TOP2 inhibitor, a CDK4/6 inhibitor, a MEK inhibitor, and a pan-CDK inhibitor) based, at least in part, on the absence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. For example, a mammal having prostate cancer can be identified as being likely to respond to one or more TOP2 inhibitors, one or more CDK4/6 inhibitors, one or more MEK inhibitors, and/or one or more pan-CDK inhibitors based, at least in part, on the absence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample obtained from the mammal.
Any appropriate sample from a mammal (e.g., a human) having prostate cancer can be assessed as described herein (e.g., for the presence or absence of an increased level of expression of one or more polypeptides). In some cases, a sample can be a biological sample. In some cases, a sample can contain one or more cancer cells. In some cases, a sample can contain one or more biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and/or metabolites). Examples of samples that can be assessed as described herein include, without limitation, tissue samples (e.g., prostate tissue samples or prostate cancer tissue biopsies), fluid samples (e.g., whole blood, serum, plasma, urine, and saliva), and cellular samples (e.g., samples containing circulating cancer cells). A sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, a sample can be a processed sample (e.g., an embedded sample such as a paraffin or OCT embedded sample). In some cases, one or more biological molecules can be isolated from a sample. For example, nucleic acid (e.g., DNA and RNA such as messenger RNA (mRNA)) can be isolated from a sample and can be assessed as described herein. In some cases, one or more polypeptides can be isolated from a sample and can be assessed as described herein.
When treating a mammal (e.g., a human) having prostate cancer and identified as being likely to respond to one or more anti-androgen agents as described herein (e.g., based, at least in part, on the absence of an increased level of expression of one or more polypeptides), the mammal can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, or more) anti-androgen agents. For example, a mammal having cancer and identified as lacking an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer one or more anti-androgen agents. For example, a mammal having prostate cancer and identified as lacking an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer one or more anti-androgen agents. Examples of anti-androgen agents include, without limitation, leuprolide (e.g., LUPRON DEPOTR and ELIGARDR), goserelin (e.g., ZOLADEXR), triptorelin (e.g., TRELSTARR), histrelin (e.g., VANTASR), degarelix (e.g., FIRMAGONR), abiraterone (e.g., ZYTIGAR), ketoconazole (e.g., NIZORAL R), flutamide (e.g., EULEXINR), bicalutamide (e.g., CASODEXR), nilutamide (e.g., NILANDRONR), enzalutamide (e.g., XTANDIR), apalutamide (e.g., ERLEADAR), and darolutamide (e.g., NUBEQAR).
In some cases, a mammal (e.g., a human) having prostate cancer and identified as being likely to respond to one or more anti-androgen agents as described herein (e.g., based, at least in part, on the absence of an increased level of expression of one or more polypeptides) can undergo a surgical hormone therapy (e.g., in addition to or as an alternative to being administered one or more anti-androgen agents). For example, a mammal having prostate cancer and identified as lacking an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can undergo an orchiectomy (removal of the testicles).
When treating a mammal (e.g., a human) having prostate cancer and identified as being unlikely to respond to one or more anti-androgen agents as described herein (e.g., based, at least in part, on the presence of an increased level of expression of one or more polypeptides), the mammal can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, or more) alternative cancer treatments (e.g., one or more cancer treatments that are not an anti-androgen agent). For example, a mammal having prostate cancer and identified as having the presence of an increased level of expression of CCNA2 polypeptides, CCNB1 polypeptides, CCNB2 polypeptides, PRC1 polypeptides, SMC2 polypeptides, DLGAP5 polypeptides, ECT2 polypeptides, FBXO5 polypeptides, CDK 1 polypeptides, NCAPG polypeptides, and/or KIF4A polypeptides in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer one or more alternative cancer treatments that are not anti-androgen agents. Examples of alternative cancer treatments that are not an anti-androgen agent include, without limitation, administering one or more cancer drugs (e.g., chemotherapeutic agents, targeted cancer drugs, and immunotherapy drugs) other than an anti-androgen agent to a mammal in need thereof. Examples of cancer drugs that are not an anti-androgen agent and that can be administered to a mammal having prostate cancer and identified as being unlikely to respond to an anti-androgen agent include, without limitation, docetaxel (e.g., TAXOTERER), cabazitaxel (e.g., JEVTANAR), mitoxantrone (e.g., NOVANTRONER), doxorubicin (e.g. ADRIAMYCINR, CAELYXR, and MYOCETR) palbociclib (e.g., IBRANCER), ribociclib (KISQALIR), abemaciclib (VERZENIOR), PD-0325901 (mirdametinib), and PHA-793887, and combinations thereof. In some cases, an alternative cancer treatment can include surgery. Examples of surgeries that can be performed on a mammal having prostate cancer include, without limitation, radical prostatectomy (removal of the prostate gland). In some cases, an alternative cancer treatment can include radiation treatment. In some cases, an alternative cancer treatment can include prostate tissue ablation. Examples of ablative therapies that can be performed on a mammal having prostate cancer to treat the mammal include, without limitation, freezing prostate tissue (e.g., cryoablation or cryotherapy) and heating prostate tissue.
When treating a mammal (e.g., a human) having prostate cancer and identified as being unlikely to respond to one or more anti-androgen agents as described herein (e.g., based, at least in part, on the presence of an increased level of expression of one or more polypeptides), the mammal can be administered or instructed to self-administer (a) one or more (e.g., one, two, three, four, five, or more) anti-androgen agents and (b) one or more (e.g., one, two, three, four, five, or more) agents that can sensitize prostate cancer to one or more anti-androgen agents. For example, a mammal having prostate cancer and identified as being unlikely to respond to one or more anti-androgen agents as described herein can be administered an anti-androgen agent (e.g., abiraterone) and also can be administered one or more agents that can sensitize a prostate cancer to treatment with one or more anti-androgen agents. In some cases, an agent that can sensitize a prostate cancer to one or more anti-androgen agents can be a TOP2 inhibitor (e.g., a TOP2A inhibitor). In some cases, an agent that can sensitize a prostate cancer to one or more anti-androgen agents can be a CDK 4/6 inhibitor. Examples of agents that can sensitize a prostate cancer to one or more anti-androgen agents (e.g., abiraterone) include, without limitation, mitoxantrone (e.g., NOVANTRONE®), doxorubicin (e.g. ADRIAMYCIN®, CAELYX®, and MYOCET®) palbociclib (e.g., IBRANCE®), ribociclib (KISQALI®), abemaciclib (VERZENIO®), PD-0325901 (mirdametinib), PHA-793887, teniposide, daunorubicin, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331. In some cases, the one or more anti-androgen agents can be administered together with the one or more agents that can sensitize a prostate cancer to one or more anti-androgen agents. In some cases, the one or more anti-androgen agents can be administered independent of the one or more agents that can sensitize a prostate cancer to one or more anti-androgen agents. When the one or more anti-androgen agents are administered independent of the one or more agents that can sensitize a prostate cancer to one or more anti-androgen agents, the one or more agents that can sensitize a prostate cancer to one or more anti-androgen agents can be administered first, and the one or more anti-androgen agents administered second, or vice versa.
When treating a mammal (e.g., a human) having prostate cancer by administering one or more (e.g., one, two, three, four, five, or more) anti-androgen agents, the mammal also can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, or more) steroids (e.g., a corticosteroid), where the one or more cancer treatments are effective to treat the cancer within the mammal. Examples of steroids that can be administered to a mammal having prostate cancer together with one or more anti-androgen agents can include, without limitation, predisone, prednisolone, methylprednisolone, dexamethasone, and combinations thereof. In some cases, the one or more steroids can be administered together with the one or more anti-androgen agents. In some cases, the one or more steroids can be administered independent of the one or more anti-androgen agents. When the one or more steroids are administered independent of the one or more anti-androgen agents, the one or more steroids can be administered first, and the one or more anti-androgen agents administered second, or vice versa.
In some cases, when treating a mammal (e.g., a human) having prostate cancer as described herein, the treatment can be effective to treat the cancer. For example, the number of cancer cells present within a mammal can be reduced using the materials and methods described herein. In some cases, the size (e.g., volume) of one or more tumors present within a mammal can be reduced using the methods and materials described herein. For example, the methods and materials described herein can be used to reduce the size of one or more tumors present within a mammal having prostate cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the size (e.g., volume) of one or more tumors present within a mammal does not increase.
In some cases, when treating a mammal (e.g., a human) having prostate cancer as described herein, the treatment can be effective to improve survival of the mammal. For example, the methods and materials described herein can be used to improve overall survival. For example, the methods and materials described herein can be used to improve disease-free survival (e.g., relapse-free survival). For example, the methods and materials described herein can be used to improve progression-free survival. For example, the methods and materials described herein can be used to improve the survival of a mammal having prostate cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to improve the survival of a mammal having prostate cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years).
In some cases, when treating a mammal (e.g., a human) having prostate cancer as described herein, the treatment can be effective to reduce one or more symptoms of the cancer. Examples of symptoms of prostate cancer include, without limitation, trouble urinating, decreased force in the stream of urine, blood in the urine, blood in the semen, bone pain, losing weight without trying, and erectile dysfunction. For example, the materials and methods described herein can be used to reduce one or more symptoms within a mammal having prostate cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
This Example describes the identification of genes that are differentially expressed between prostate cancers that are abiraterone (Abi) responders and prostate cancers that are Abi non-responders. This Example also describes drugs that can be used to sensitize Abi-resistant prostate cancers to Abi treatment.
All patient specimens were collected as part of the PROMOTE study as described elsewhere (see, for example, Champoux. Annu. Rev. Biochem., 70, 369-413 (2001); and Kellner et al., Lancet Oncol., 3, 235-243 (2002)). Two pathology confirmed mouse PDX (patient-derived xenograft) models were generated from AA/P non-responder bone metastatic tumor biopsy tissues of metastatic castration-resistant prostate cancer (mCRPC), with one (MC-PRX-01) from baseline pretreated samples and one (MC-PRX-06) after 12-weeks of AA/P treatment as described elsewhere (see, for example, Kohli et al., PLOS One, 10, e0145176 (2015); and Wang et al., Ann. Oncol., 29, 352-360 (2018)). These two models were used to test the in vivo tumor response to abiraterone±doxorubicin/mitoxantrone. Pieces of tumor (about 20 mg pieces having about 3-4 mm per cubed side) mixed with matrigel were implanted subcutaneously into 6-8 weeks old male CB17 NOD-SCID mice (Charles River Laboratories, Raleigh, North Carolina). After the tumor reached 100 mm3, mice were randomized to 6 groups. Each group of mice (n=4 or 5) was administered, for 28 consecutive days, a vehicle control daily, 200 mg/kg abiraterone (dissolved in 5% benzyl alcohol and 95% safflower oil) daily, 2 mg/kg doxorubicin (saline solution) once every 14 days, 0.45 mg/kg mitoxantrone (saline solution) once every week, combination of Abi plus doxorubicin, or combination of Abi plus mitoxantrone. Body weight and tumor volumes (width2×length/2) were measured two to three times per week with a digital caliper, and the average tumor volumes were determined. At the end of the treatment period, mice were euthanized, and the tumors were removed, dissected, and frozen at −80° C. for further analysis.
PDX tumor dissociation, tumor cell isolation and organoid formation were done as described elsewhere (see, for example, Yu et al., J. Clin. Invest., 128, 2376-2388 (2018)). The tumor cell dissociation kit, cell strainer, and mouse cell depletion kit were purchased from Miltenyi Biotec. Harvested tumors were dissected into 2 mm sections, and were incubated with 5 mL of the human Tumor Dissociation enzyme mix. The tumor tissue was digested gently on a MACS Dissociator, and was passed through a 70 μm and a 40 μm MACS SmartStrainer sequentially. After centrifugation and washing with precooled washing buffer, mouse cells were removed using a Mouse Cell Depletion Purification Kit according to the manufacturer's protocol.
To culture tumor organoids, 1.5-2×104 live cells were cultured in 96 well NanoCulture Plate (NCP) (Scivax Corp) in 100 μL modified MEF medium (Phenyl-red free DMEM supplemented with 10% charcoal-stripped FBS, 1% glutamax, 1% sodium pyruvate, 1% nonessential amino acids, 1% penicillin-streptomycin (Life Technologies, Grand Island, NY)), supplemented with 5 μM Y27632 ROCK inhibitor (Tocris Bioscience, Bristol, United Kingdom) and 50 nM pregnenolone. The medium was replaced every 3-5 days. Tumor organoids were allowed to grow for 3 to 7 days before drug testing. Organoids were then treated with a variable concentration of abiraterone with or without the indicated concentration of mitoxantrone, palbociclib, PD-0325901 or PHA-793887 (MedChemExpress) for 5 days before viability was examined by 3D cell titer kit (Promega, Madison, WI). Solvent was used as the control.
Drugs potentially reversing abiraterone resistant expression profiles were identified using the Enrichr portal. Significantly up- or down-regulated genes were identified using transcriptomic data of PROMOTE patients (see, for example, Wang et al., Ann. Oncol., 29, 352-360 (2018)) and the 5 PDX models from this study. Those genes were used as input against LINCS L1000 Chem Pert down/up database using the Enrichr portal (amp.pharm.mssm.edu/Enrichr/) as described elsewhere (see, for example, Kuleshov et al., Nucleic Acids Res., 44, W90-97 (2016); and Chen et al., BMC Bioinformatics, 14, 128 (2013)). Search returned signatures with false discovery rate (FDR) adjusted p-values≤0.05 were downloaded. Signatures of each drug were then combined by either the total number of significant signatures or weighted mean ranks (calculated using the formula as below). Target genes modulated by each drug were pooled from all significant signatures.
The Rank Score was calculated as following: Significant signatures were reverse ranked by p-values (signature with smallest p-value received largest rank), and weighted mean ranks (referred as Rank Score) for each drug i with total number of n significant signatures were calculated by
where weightsi,j=2−j+1 for the jth signature ordered by smallest p-values for drug i.
The Rank Score was calculated so that only the first few most significant signatures for each drug were considered.
Transcriptomic Data from PROMOTE, SU2C and TCGA Cohorts
Baseline transcriptomic data and clinical data of the PROMOTE cohort were published elsewhere and can be accessed through dbGAP (phs001141.v1.p1) (see, for example, Champoux, Annu. Rev. Biochem., 70, 369-413 (2001)). 68 samples that passed quality control were included in the study, including 46 bones, 13 lymph nodes and 9 other metastatic sites. Raw count of PROMOTE RNAseq data was normalized by conditional quantile normalization (CQN) method and log 2 transformed, as described (see, for example, Hansen et al., Biostatistics, 13, 204-216 (2012)). The drug response was measured by 12 weeks progression determined by composite scores as described earlier, as well as overall survival (46 deceased, 22 living) and time to treatment change (TTTC, 53 treatment changed, 15 not changed).
RNAseq data of Stand Up 2 Cancer (SU2C) cohort data was downloaded from cBioPortal (cbioportal.org,) as log 2 transformed RPKM values. Patients with treatment naïve target capture RNAseq data, overall survival, and having received Abi treatment were included. One sample (TP_2079_Tumor) was excluded due to the low reads. The final analyzable cohort included 53 samples including 23 bones, 22 lymph nodes, and 8 other metastatic sites. For treatment outcome, overall survival (32 deceased, 21 living) was used. TCGA pan-cancer cohort batch effects normalized mRNA data was downloaded from UCSC Xenabrowser (xenabrowser.net) as log 2 transformed RPKM values. Progression-free survival was used as the outcome. Prostate Cancer (93 progressed, 403 non-progressed), breast cancer (147 progressed, 951 non-progressed), cervix cancer (72 progressed, 234 non-progressed), and colon cancer (84 progressed, 204 non-progressed) were included.
Subgroups of patients who might be sensitive to the 4 top candidate drugs were identified by the k-means clustering method calculated from the z-score transformed 11 gene expression. The optimal number of clusters were determined by the elbow method (
Prognosis prediction value was examined by the Cox proportional hazard model by either univariate or multivariate including various gene panels and log 10(PSA). The 11 gene panel was input into the model as a binary variable representing “high expression” vs “low expression” groups. CCP score, AR score NEPC scores, copy number variation, and mutation calls of PROMOTE cohort were determined as described elsewhere (see, for example, Wang et al., Ann. Oncol., 29, 352-360 (2018)). AR activity score and NEPC scores for SU2C cohorts were downloaded from cBioPortal and CCP scores were calculated by sum up the z-score transformed expression of CCP genes, as described elsewhere (see, for example, Wang et al., Ann. Oncol., 29, 352-360 (2018)). COX modeling, Log-likelihood ratio test and Akaike information criterion (AIC) calculation were performed using a survival package from R software.
22Rv1 and LNCaP cells purchased from ATCC were routinely cultured in RPMI1640 medium (Gibco, Grand Island, NY) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA) and 1% Pen-Strep. To develop Abi resistant cell line, cells were maintained in the medium supplemented with 5 μM of abiraterone (Sellect Chemicals, Houston, TX) for 3 months until the viability reached over 95%. Abi resistant cells (LNCaP-AbiRes or 22Rv1-AbiRes) were compared with parental cells for viability and gene expression after Abi and other treatments.
Forty eight hours before abiraterone treatment, cell was seeded in phenol red-free RPMI (Gibco, Grand Island, NY) supplemented with 10% charcoal stripped FBS (Thermo Fisher Scientific, Waltham, MA), and 50 nM pregnenolone was added after 24 hours. Cells were then treated with abiraterone at various doses with or without mitoxantrone, palbociclib, PD-0325901 or PHA-793887 at indicated concentrations for 3 days before viability being examined by Cyquant direct assay (Thermo).
qRT-PCR
Total RNA for qRT-PCR was extracted from tumor organoids or cell lines using Quick-RNA MiniPrep Kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. qRT-PCR was performed using the Power SYBR® Green RNA-to-CT 1-Step Kit (Life Technologies, Grand Island, NY) and QuantiTect® (QIAGEN, Germantown, MD) or PrimeTime® (IDT, Inc., Coralville, Iowa) pre-designed qPCR primers (IDT Coralville, IA). Gene expression analyses were performed using ΔΔCt method, and β-actin was used as the internal reference. Three independent experiments were performed. Primer sequences are in Table 1.
RNAseq of PDX tumor was performed by ACGT, Inc. Total RNA from 5 PDX models (MC-PRX-01, MC-PRX-03, MC-PRX-04, MC-PRX-07, MC-PRX-08) was extracted using the RNeasy Plus Mini kit (QIAGEN, Germantown, MD), per manufacturer's instructions. At least 3 tumors were included for each PDX model from various passages of mice based on tumor availability and quality. mRNA libraries were enriched using a NEXTflex™ Rapid Directional qRNASeq™ Kit. Quantity and quality of RNA libraries were evaluated by Qubit fluorometry and Agilent 2100 Bioanalyzer. Individual libraries were pooled in equimolar ratios, and were run on HiSeq 4000 system (2×150 paired end, Illumina, San Diego, CA).
Low quality were trimmed by Trim Galore, and reads of mouse origin were removed by BBsplit. Filtered reads were aligned to hg19 human reference genome using Hisat2 with average mapping rate is 93%. Raw counts were then called by HTSeq excluding non-unique mapped reads. One sample (MC-PRX-08.1) was excluded due to poor consistency within replicates. Differential expression analysis was performed between 1 model derived from AA/P responder (MC-PRX-04) and 4 models derived from AA/P non-responders (MC-PRX-01, MC-PRX-03, MC-PRX-07, MC-PRX-08) EdgeR. Genes differentiate by fold change ≥2 and FDR≤0.05 were considered significant.
Four Drugs were Identified as Candidate Drugs to Reverse Abi Resistance Expression Phenotype
Drug identification workflow was illustrated in
The database was searched using DEGs between AA/P non-responder and responders in bone metastasis samples, which comprises 70% of the samples of PROMOTE cohort. Using 103 upregulated genes, 689 drugs were identified with at least one signature passing FDR 0.05, with a mean of 7.3 and a median of 2 signatures per drug (
Four PDX models (MC-PRX-01, MC-PRX-03, MC-PRX-07, MC-PRX-08) from AA/P non-responders and one PDX model (MC-PRX-04) from AA/P responder were successfully established using the AA/P treatment naïve metastatic biopsies (visit 1) from the PROMOTE patients. Differential expression analysis of the RNAseq data from AA/P non-responders and responder PDXs identified 377 upregulated and 763 downregulated genes in PDX from AA/P non-responders (FDR≤0.05 and fold change ≥2) (Table 5). Volcano plots for differentially expressed genes (DEG) were presented in
The gene targets potentially modulated by these four drugs were examined from the L1000 signatures. These drugs shared highly similar profile of gene expression modulation. Using patient's RNAseq data as input, 89 DEGs are modulated by at least one of the four drugs, among them, 50 genes were shared among all drugs (
To examine whether the four identified drugs can sensitize abiraterone treatment in prostate cancer, Abi resistant prostate cancer cell models in 22Rv1 and LNCaP (22Rv1-AbiRes and LNCap-AbiRes) were generated. The resistant phenotypes were tested using the cytotoxicity assay (
Expression of the 11 potential drug targeting genes was further validated using qRT-PCR in parental and Abi-resistant prostate cancer lines as well as in the PDX derived organoids. As shown in
Among the four drugs, mitoxantrone, is currently the only FDA approved drug for CRPC patients. It was the most potent in the cytotoxicity experiments and showed more significant effects on the 11 gene suppression (
The expression of the 11 drug targeting genes in the PDX tumors harvested after various treatments was further examined. Expression of the 11 genes was significantly downregulated after drug treatment in both mitoxantrone and doxorubicin treated tumors (
The 11 genes were upregulated in AA/P non-responders' tumor samples and PDX models derived from AA/P non-responders (
Unsupervised machine learning by k-means clustering analysis was performed using the 11 genes on 68 PROMOTE baseline patient samples from all biopsy sites (
A second independent cohort, the Stand Up To Cancer (SU2C) was also analyzed (see, for example, Abida et al., Proceedings of the National Academy of Sciences 116, 11428-11436 (2019)). The trial has two treatment arms, enzalutamide and abiraterone. To best resemble the PROMOTE cohort, the Abi-naïve biopsy samples from the Abi treatment arm that had overall survival data were used (53 samples, Table 7). Using the 11 gene panel and clustering strategy that was applied in the PROMOTE cohort, the expression data of the SU2C cohort can also be clustered into high and low expression subgroups. The patients in high expression cluster exhibited worse survival outcome as compared with the low expression clusters with a p-value of 0.0208 (
The effect of the gene panel in the context of other genomic alterations and clinical variables in the PROMOTE samples was examined using the Spearman correlation of the expression of the 11 genes with a number of clinical variables, including PSA and testosterone levels, as well as three widely used gene panels or scores that are known to be associated with prostate cancer progression, including AR activity score (20 genes) (see, for example, Hieronymus et al., Cancer Cell 10, 321-330 (2006)), cell cycle progression (CCP) (31 genes) (see, for example, Cuzick et al. The Lancet Oncology 12, 245-255 (2011)), and neuroendocrine prostate cancer (NEPC) (70 genes) (see, for example, Beltran et al., Nat. Med., 22, 298-305 (2016)). The 11 genes identified here were highly correlated with the CCP score (
The prognostic prediction values of the gene panels were further analyzed by COX proportional hazard models. Univariate analysis identified log-PSA as the only clinical variable associated with prognosis. Expression of AR-V7, CCP score and NEPC score were also identified to be significantly associated with overall survival as described elsewhere (see, for example, Cuzick et al., The Lancet Oncology, 12, 245-255 (2011); Beltran et al., Nat. Med., 22, 298-305 (2016); Hu et al., Cancer Res., 69, 16-22 (2009); and Sommariva et al., European Urology, 69, 107-115 (2016)). The 11 gene expression clusters, compared with other gene panels tested here, are more significantly associated with overall survival (
Similar analyses were performed using the data from the Abi-treated SU2C cohort. As shown in
All patient specimens were collected as part of the PROMOTE study as described elsewhere (see, for example, Champoux. Annu. Rev. Biochem., 70, 369-413 (2001); and Kellner et al., Lancet Oncol., 3, 235-243 (2002)). Briefly, pathology confirmed metastatic tumor biopsy tissues of mCRPC was renal capsule xenografted and 25 mg testosterone pellet was subcutaneously implanted into 6-8 weeks old male CB17 NOD-SCID mice (Jackson laboratories, Bar Harbor, Maine; Charles River Laboratories, Raleigh, North Carolina) and observed for at least 6 months or till the tumor reached 1.0-1.5 cm at maximal length, when sub-xenografts were expanded by subcutaneous implantation with harvested PDX tumor mixed with Matrigel (Corning, Corning, New York) with no exogenous testosterone supplied. Early generation PDX tumors were collected for cryo-storage and next-generation sequencing. Pathology confirmed PDX model generated from AA/P non-responder bone metastatic tumor biopsy tissues were employed to test the in vivo tumor response to abiraterone+Mito/Dox/Palb/PHA. After PDX tumor reached approximately 100 mm3, mice were randomized to 6 groups. Each group of mice (n=5) was administered, for 35 consecutive days, with vehicle control daily, 200 mg/kg abiraterone (dissolved in 5% benzyl alcohol and 95% safflower oil) per oral 5 days/week, 150 mg/kg palbociclib (50 mmol/L, Sodium lactate, 5% Tween80) per oral 5 days/week, 20 mg/kg PHA-793887 (5% dextrose, 5% DMSO) intravenous three times per week, a combination of Abi plus palbociclib, or a combination of Abi plus PHA-793887. Body weight and tumor volumes (width2×length/2) were measured two to three times per week with a digital caliper, and the average tumor volumes were determined. At the end of the treatment period, mice were euthanized and the tumors were removed, dissected and frozen at −80° C. for further analysis.
While both PHA and Palb inhibited tumor growth, Palb appeared to be effective as single treatment, while PHA-alone did not exhibit statistically significant inhibition at tested dosage, but did appear to sensitize the tumor to Abi treatment (
DKFZ early onset prostate cancer cohort was downloaded from the cBioPortal as log 2 transformed RPKM values. Patients with RNAseq data and biochemical relapse (BCR) was included. The final analyzable cohort included 105 samples with 24 relapsed and 81 non-relapsed (Table 8).
Using the 11 gene panel and clustering strategy that was applied to the PROMOTE dataset (see, Example 1), the expression data of the SU2C cohort can also be clustered into high and low expression subgroups, and that patients in high expression cluster also exhibited worse overall survival as compared to the low expression cluster (p-value=0.0208). When including samples in the enzalutamide treatment arm (9 additional samples with available data), only one more sample was clustered into high-expression subgroup and survival benefits were essentially the same as abiraterone-arm only (
In order to examine whether the panel also has a prognostic value of disease relapse in the primary tumor, the German Cancer Research Center (DKFZ) early-onset prostate cancer cohort was further examined. Progression-free survival (PFS) was used as the clinical outcome for the TCGA cohort and biochemical relapse (BCR) for the DKFZ cohort. The results also showed that the patients with high expression or in the high expression subgroup had a worse PFS outcome in the TCGA cohort (p-value=6.78×10−6) and a worse BCR outcome in the DKFZ cohort (p-value=6.55×10−5) (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 63/129,393, filed on Dec. 22, 2020. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/064539 | 12/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63129393 | Dec 2020 | US |
