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Supplemental Figure 1 (fig. S1). Representative whole body NaF-18 PET scan images after 44 days of (A) vehicle-treated and (B) cabozantinib-treated animals. Supplemental Figure 2 (fig. S2). Effect of cabozantinib on PDX tumor growth, animal weight, and survival. Supplemental Figure 3 (fig. S3). Representative images of viable islets of tumor cells in (A) Patients; (B) MDA PCa-118b grown intrafemurally; and (C) MDA PCa- 118b grown subcutaneously. Supplemental Figure 4 (fig. S4). Time-dependent effect of cabozantinib on expression and phosphorylation of primary and MET downstream targets. Supplemental Figure 5 (fig. S5). Relative expression of c-met mRNA in NT and knockdown cells as determined by qRT-PCR. Supplemental Figure 6 (fig. S6). MET expression and activity in NT and knockdown tumors. Supplemental Figure 7 (fig. S7): Quantitation of CD31 staining on bone tumors. Supplemental Figure 8 (fig. S8). Effects of cabozantinib on bone remodeling markers in the phase-2 clinical trial and growth and differentiation of primary osteoblasts. Supplemental Figure S9 (fig S9). Effect of cabozantinib on bone turnover in the Phase 2 trial and PDX grown intrafemurally. Supplemental Figure 10 (fig. S10). Quantitation of phospho-histone H3 staining.

Supplemental Table 1. Summary of the clinical and histopathologic characteristics of Merkel cell carcinoma patients. Supplemental Table 2. Summary of antibodies used in this study. Supplemental Table 3. Correlation between tumor associated immune infiltrate and co-localized expression of CD31 and B7-H3 in Primary MCCs.

Purpose: We performed parallel investigations in cabozantinib-treated patients in a phase II trial and simultaneously in patient-derived xenograft (PDX) models to better understand the roles of MET and VEGFR2 as targets for prostate cancer therapy.Experimental Design: In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR2, bone turnover, angiogenesis, and resistance were examined.Results: In responsive patients and PDX, islets of viable pMET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice nor did it affect cabozantinib-induced growth inhibition but did lead to induction of FGFR1. Inhibition of VEGFR2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR2-negative vessel. Reduction of bone turnover was observed in both cohorts.Conclusions: Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic castrate-resistant prostate cancer (CRPC), but inhibition of VEGFR2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents. Clin Cancer Res; 22(1); 107–21. ©2015 AACR.

Supplemental Figure 1. Dynamic range of co-localized expression of CD31 and B7-H3 in primary Merkel cell carcinoma. Each CD31+ and B7-H3+ cell centroid was tabulated as a series of (X, Y) coordinates (cell centroids). The frequency of their respective overlap for each of the 52 primary MCCs was calculated within a given distance as indicated: blue boxes show G-function colocalization index applying a 1 pixel (1.57 �m) minimum distance of overlapped expression as our cutoff, and green boxes show G-function colocalization index applying a 2 pixel (3.14 �m) minimum distance of overlapped expression as our cutoff.

Purpose:Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy whose pathogenesis and prognosis are related to the integrity of the host immune system. Despite promising clinical responses to immune-checkpoint blockade, response and resistance remain unpredictable, underscoring a critical need to delineate novel prognostic biomarkers and/or therapeutic targets for this disease.Experimental Design: Expression of immune-regulatory markers (PD-L2, B7-H3, B7-H4, IDO-1, ICOS, TIM3, LAG3, VISTA, and OX-40) was assessed using singlet chromogenic IHC in 10 primary MCCs. Multiplex immunofluorescence quantified CD31 and B7-H3 expression in 52 primary and 25 metastatic MCCs. B7-H3 and CD31 expressions were tabulated as a series of independent (X,Y) cell centroids. A spatial G-function, calculated based on the distribution of distances of B7-H3+ (X,Y) cell centroids around the CD31+ (X,Y) cell centroids, was used to estimate a colocalization index equivalent to the percentage of CD31-positive cell centroids that overlap with a B7-H3–positive cell centroid.Results:Primary and metastatic MCCs exhibit a dynamic range of colocalized CD31 and B7-H3 expression. Increasing colocalized expression of B7-H3 with CD31 significantly associated with increased tumor size (P = 0.0060), greater depth of invasion (P = 0.0110), presence of lymphovascular invasion (P = 0.0453), and invasion beyond skin (P = 0.0428) in primary MCC. Consistent with these findings, increasing colocalized expression of B7-H3 and CD31 correlated with increasing vascular density in primary MCC, but not metastatic MCC.Conclusions:Our results demonstrate that colocalized expression of B7-H3/CD31 is a poor prognostic indicator and suggest therapies targeting B7-H3 may represent an effective approach to augmenting immune-activating therapies for MCC.

Supplemental Figure 2. Correlation between B7-H3/CD31 G-function co-localization index in MCPyV-positive MCCs. Plots showing the correlation B7-H3/CD31 G-function co-localization index for each primary MCPyV-positive MCC plotted against tumor size (A), depth of invasion (B), invasion beyond skin (C) and metastasis beyond SLN (D). G-function calculated according to a 1 pixel (1.57 �m) radius.

Supplemental Figure 3. Correlation between B7-H3/CD31 G-function co-localization index in MCPyV-negative MCCs. Plots showing the correlation B7-H3/CD31 G-function co-localization index for each primary MCPyV-negative MCC plotted against the frequency of associated malignancies (A) and lymphovascular invasion (B). G-function calculated according to a 1 pixel (1.57 �m) radius.

supplementary materials and methods, supplementary tables and supplementary figure legends Supplemental Table 1: Patient Characteristics. Supplemental Table 2: Targeting sequences for c-met. Supplemental Table 3: Antibodies and Sources. Supplemental Table 4: Oligonucleotides for PCR Amplification of Mouse Alkaline Phosphatase and Osteonectin.

Supplemental Figure 4. Correlation between B7-H3/CD31 G-function co-localization index and vascular density. Plots showing the correlation B7-H3/CD31 G-function co-localization index plotted against vascular density for the whole cohort of primary MCC (vascular density tabulated as the average of the regions of interest in each of the 52 tumors).

Despite advancements in treatment, prostate cancer (PCa) remains the second leading cause of death among men. Neuroendocrine prostate cancer (NEPC) represents one of the most lethal forms of PCa and lacks life-prolonging treatment. Here we identified histone lysine demethylase KDM4A as a driver in NEPC progression and an effective therapeutic target. KDM4A mRNA and protein are overexpressed in human and mouse NEPC compared to adenocarcinoma. Knockdown or knockout of KDM4A in NEPC cell lines suppressed cancer cell growth in vitro and in vivo. Importantly, the inactivation of Kdm4a in a genetically engineered mouse model of prostate cancer reduces tumor burden, reduces the incidence of NEPC, and prolongs overall survival. Mechanistically, KDM4A directly regulates the transcription of MYC, which is hyper-activated in human and mouse NEPC. Furthermore, a potent pan-KDM4 inhibitor QC6352 significantly reduces NEPC cell growth in vitro and in vivo. Taken together, we demonstrate that KDM4A promotes NEPC progression through regulation of MYC expression and targeting KDM4A can be an effective therapeutic strategy for NEPC. Citation Format: Celia Sze Ling Mak, Ming Zhu, Xin Liang, Feng Wang, Anh G. Hoang, Xinzhi Song, Peter Shepherd, Derek Liang, Jessica Suh, Jiwon Park, Miao Zhang, Eric Metzger, Roland Schule, Abhinav K. Jain, Ellen Karasik, Barbara A. Foster, Min Gyu Lee, Paul Corn, Christopher J. Logothetis, Ana Aparicio, Nora Navone, Patricia Troncoso, Jianhua Zhang, Sue-Hwa Lin, Guocan Wang. KDM4A promotes NEPC progression through regulation of MYC expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4760.

Neuroendocrine prostate cancer (NEPC) represents one of the most lethal forms of prostate cancer (PCa) and lacks life-prolonging treatment. The incidence of NEPC is increased due to the widespread use of AR pathway inhibitors (ARPIs) in the treatment of non-metastatic CRPC and hormone-sensitive metastatic tumors. Here, we identified histone lysine demethylase KDM4A as a key player in NEPC progression and an effective therapeutic target. We found that KDM4A mRNA and protein are overexpressed in human and mouse NEPC compared to prostate adenocarcinoma. Knockdown (KD) or knockout (KO) ofKDM4Ain NEPC cell lines suppressed cancer cell growthin vitroandin vivo. Mechanistically, we found that KDM4A promotes NEPC progression, in part, through direct transcriptional regulation ofMYC. We showed thatMYCis hyper-activated in human and mouse NEPC.KDM4AKD led to suppression of MYC signaling.MYCKD or inhibition profoundly suppressed NEPC cell proliferation. Furthermore, a potent pan-KDM4 inhibitor QC6352 significantly reduced NEPC cell growthin vitroandin vivo. Taken together, we demonstrated that KDM4A is an important regulator of NEPC progression and targeting KDM4A may potentially be an effective therapeutic strategy for NEPC.SignificanceNeuroendocrine prostate cancer (NEPC) is a highly aggressive prostate cancer subtype that is resistant to potent androgen receptor pathway inhibitors (ARPIs) and currently lacks effective therapeutic options. Histone lysine demethylase KDM4A is an important epigenetic regulator of gene expression in development and cancer. In this study, we show that KDM4A is highly expressed in NEPC and is required for NEPC proliferation, anchorage-independent growth, andin vivogrowth, which is in part mediated through the regulation of MYC expression. Importantly, we demonstrate that inhibition of KDM4A significantly impairs NEPC growth in preclinical models. Thus, our findings provide valuable insights into the molecular mechanisms underlying NEPC progression and offer a rationale for clinical trials with KDM4 inhibitor in NEPC patients.

BACKGROUND: The unmet need for predictive biomarkers emerged from the unpredictable pattern of response to androgen signaling inhibition in metastatic castration-resistant prostate cancer (mCRPC). Here, we report on the testing of a previously identified candidate androgen signaling signature associated with response to androgen signaling inhibition. PATIENTS AND METHODS: We report on the outcome of the first module of a phase II trial on Abiraterone acetate (AA) followed by combination with dasatinib or sunitinib. Bone marrow biopsies (BMB) with matched bone marrow aspirate and blood samples were collected at baseline and upon progression. Endpoints included assessment of a prespecified molecular signature consisting of nuclear androgen receptor (AR) overexpression, cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17) expression, and AR C-/N-terminal expression ratio of ≥0.8 by immunohistochemistry (IHC) in patients with benefit versus primary resistance to AA (i.e. progression within 4 months). Tumor markers also included v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), splice variant ARV7 by IHC and steroids by Liquid chromatography-tandem mass spectrometry. RESULTS: Out of 170 patients accrued from 03/2011 to 02/2015, 44 (26%) were primary resistant to AA. 48 patients had tumor infiltrated BMB at baseline. Pretreatment androgen signaling signature was linked to benefit from AA (p

Methylthioadenosine phosphorylase, an essential enzyme for the adenine salvage pathway, is often deficient (MTAPdef) in tumors with 9p21 loss and hypothetically renders tumors susceptible to synthetic lethality by antifolates targeting de novo purine synthesis. Here we report our single arm phase II trial (NCT02693717) that assesses pemetrexed in MTAPdef urothelial carcinoma (UC) with the primary endpoint of overall response rate (ORR). Three of 7 enrolled MTAPdef patients show response to pemetrexed (ORR 43%). Furthermore, a historic cohort shows 4 of 4 MTAPdef patients respond to pemetrexed as compared to 1 of 10 MTAP-proficient patients. In vitro and in vivo preclinical data using UC cell lines demonstrate increased sensitivity to pemetrexed by inducing DNA damage, and distorting nucleotide pools. In addition, MTAP-knockdown increases sensitivity to pemetrexed. Furthermore, in a lung adenocarcinoma retrospective cohort (N = 72) from the published BATTLE2 clinical trial (NCT01248247), MTAPdef associates with an improved response rate to pemetrexed. Our data demonstrate a synthetic lethal interaction between MTAPdef and de novo purine inhibition, which represents a promising therapeutic strategy for larger prospective trials.

BACKGROUND Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors (“vascular zip codes”) within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα–based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m2). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients. Cancer 2015;121:2411–2421. © 2015 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. The authors report on the development of a new ligand-directed peptidomimetic (termed bone metastasis-targeting peptidomimetic-11) for interleukin-11 receptor-based human vascular targeting, including the translation from preclinical studies to a first-in-class, first-in-man clinical trial in patients with metastatic, castrate-resistant prostate cancer.

BACKGROUND Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors (“vascular zip codes”) within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα–based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m2). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients. Cancer 2015;121:2411–2421. © 2015 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.