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PDF file - 410K, Dose responses, transcriptional, biological, biochemical, and in vivo supplemental data

Supplementary Table 4: Genes down-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Methods. Supplementary Figure 1. Chemical structures of the small molecule IST5-002 analogs. Supplementary Figure 2. IST5-002 inhibits phosphorylation of Stat5 in mouse and human breast cancer cells. Supplementary Figure 3. IST5-002 does not have significant inhibitory activity against 50 kinases tested. Supplementary Figure 4. IST5-002 does not affect phosphorylation of Pak1/2. Supplementary Figure 5. IST5-002 inhibits expression of Stat5 target genes in PC cells and in CML cells. Supplementary Figure 6. IST5-002 is not generally cytotoxic as determined by lack of cell death induction in cancer cell lines established from other organs. Supplementary Figure 7. CWR22Rv1 PC cells were inoculated subcutaneously into the flanks of castrated athymic nude mice supplied with sustained-release 5alpha-dihydrotestosterone (DHT)-pellets (n=10/group, 1 tumor/mouse, 1.5 ??107 CWR22Rv1 cells per site, 1 DHT pellet/mouse). Supplementary Figure 8. IST5-0-02 induces apoptosis in CWR22Rv1 tumors grown in nude mice and in clinical PCs cultured ex vivo in organ explant cultures. Supplementary Figure 9. IST5-002 induces extensive apoptotic death of imatinib-sensitive and -resistant CML cells.

Supplementary Table 3: Genes up-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Table 5: IST5-002 regulation of known Stat5 target genes in K562 cells.

Sipuleucel-T is an autologous cellular immunotherapy for asymptomatic/minimally symptomatic metastatic castrate-resistant prostate cancer (CRPC). After disease progression, control-arm patients on three double-blind, randomized phase III sipuleucel-T trials were offered, in nonrandomized open-label protocols, APC8015F, an autologous immunotherapy made from cells cryopreserved at the time of control manufacture. These exploratory analyses evaluated potential effects on survival outcomes associated with such treatment. Of 249 control-treated patients, 165 (66.3%) received APC8015F. We explored the effects of APC8015F on the overall survival (OS; Cox regression) of control-arm patients and treatment effects of sipuleucel-T versus control adjusted for APC8015F treatment [iterative parameter estimation model (IPE)]. The median time to first APC8015F infusion was 5.2 months (range, 1.8–33.1) after randomization and 2.2 months (0.5–14.6) after progression. After disease progression, median survival was longer for APC8015F-treated versus control-only treated patients [20.0 vs. 9.8 months; HR, 0.53; 95% confidence interval (CI), 0.38–0.74; P < 0.001]; however, baseline characteristics were more favorable for APC8015F-treated patients. Multivariate regression analyses identified lactate dehydrogenase, alkaline phosphatase, hemoglobin, ECOG status, age, and number of bone metastases as potential (P < 0.1) independent predictors of postprogression survival. After adjusting for these predictors, APC8015F (HR, 0.78; 95% CI, 0.54–1.11; P = 0.17) treatment trended toward improved survival. Estimated median OS benefit for sipuleucel-T versus control adjusted for APC8015F treatment was 3.9 months if APC8015F had no effect and was 8.1 months if APC8015F was equally as effective as sipuleucel-T. Exploratory analyses indicate that APC8015F treatment may have extended patient survival, suggesting the sipuleucel-T OS advantage in CRPC may be more robust than previously estimated. Cancer Immunol Res; 3(9); 1063–9. ©2015 AACR.

Supplementary Table 2: Genes regulated by both Stat5-shRNA and IST5-002 in K562 cells, corresponding to the heatmap in Figure 2g.

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Statistical Modeling Approaches for Survival Subsequent to Disease Progression for Control-Arm Patients Including Parameter Estimates for all Model Terms.

Supplemental Table 1: Structure-Activity-Relationship analysis.

GO pathway analyses per gene cluster in Figure 6A - LNCaP and C4-2.

Supplemental Methods from Novel Actions of Next-Generation Taxanes Benefit Advanced Stages of Prostate Cancer

Gene clusters corresponding with Figure 6A - LNCaP cells.

Detail description of microarray analysis and comparison with cancer stem cell dataset, and TMA of prostate cancer patients

Figure legends corresponding with Supplemental Figure 1-6.

Dose response curves of CBTX and DCTX in LAPC4, VCaP and LNCaP-abl cells. C4-2 xenograft tumor growth response to CBTX in mice. Cleaved PARP western blot analysis of LNCaP and C4-2 cells treated with 1nM CBTX or DCTX.

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged.Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism.Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338–49)

Purpose: To improve the outcomes of patients with castration-resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individualize specific treatments for patients with CRPC. These studies compared the novel taxane cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond.Experimental design: Cabazitaxel and docetaxel were compared via in vitro modeling to determine the molecular mechanism, biochemical and cell biologic impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro and in xenograft tumors for cabazitaxel response.Results: The data herein show that (i) cabazitaxel exerts stronger cytostatic and cytotoxic response compared with docetaxel, especially in CRPC; (ii) cabazitaxel induces aberrant mitosis, leading to pyknotic and multinucleated cells; (iii) taxanes do not act through the androgen receptor (AR); (iv) gene-expression profiling reveals distinct molecular actions for cabazitaxel; and (v) tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to cabazitaxel.Conclusions: Cabazitaxel not only induces improved cytostatic and cytotoxic effects, but also affects distinct molecular pathways, compared with docetaxel, which could underlie its efficacy after docetaxel treatment has failed in patients with CRPC. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization to taxanes, which could benefit up to 50% of CRPC cases. Clin Cancer Res; 21(4); 795–807. ©2015 AACR.

Table S1, Primer sequences used for gene expression analysis. Table S2, PDE pathological characteristics.

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Supplementary Figures 1-6

Purpose:DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways.Results:Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK–mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo.Conclusions:These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.

Supplementary Data from Cyclin D1 Splice Variants: Polymorphism, Risk, and Isoform-Specific Regulation in Prostate Cancer

Gene clusters corresponding with Figure 6A - LNCaP cells.

Supplementary Figure from A Novel Role for DNA-PK in Metabolism by Regulating Glycolysis in Castration-Resistant Prostate Cancer

Bivariate flow cytometry analyses of LNCaP and C4-2 cells treated with 1nM DCTX for 16 or 48h.

GO analyses of gene clusters corresponding with Figure 6A.

Purpose:DNA-dependent protein kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer, and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. Although DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes.Experimental Design:Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacologic and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE).Results:Key findings reveal: (i) the first-in-field DNA-PK protein interactome; (ii) numerous DNA-PK novel partners involved in glycolysis; (iii) DNA-PK interacts with, phosphorylates (in vitro), and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2; (iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate; (v) DNA-PK regulates glycolysis in vitro, in vivo, and ex vivo; and (vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease.Conclusions:Findings herein unveil novel DNA-PK partners, substrates, and function in prostate cancer. DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK–mediated aggressive disease phenotypes.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1–regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src–mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723–34. ©2016 AACR.