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Supplemental Figure 1

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The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function.Significance:This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease.

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC–regulated gene expression. In AR-SV–driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer.Significance:Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995

Supplementary Figure 8 - PDF file 140K, Supplemental data demonstrating that DNAPKcs is required for DNA repair in the presence of androgen

Supplementary Figure 5,6 - PDF file 143K, Supplemental data demonstrating that DNAPKcs knockdown is maintained in growth studies and ATM expression and activity is regulated by DNAPKcs knockdown and inhibition

Supplementary Figure 1,2 - PDF file 56K, Supplemental data demonstrating that scheduling of IR and ADT treatment does not alter cell survival and DNA damage induces AR mediated gene transcription in a dose dependent fashion

Supplementary Methods - PDF file 79K, Additional materials and methods not described in main manuscript

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

Supplemental Figure 1: PLX8394 does not alter CRAF or c-Src phosphorylation; Supplemental Figure 2. Treatment of xenograft-derived tumor pieces in an ex vivo explant system; Supplemental Figure 3: Patient sample extended data; Supplemental Figure 4: Parallel ex vivo dosing and homodimerization of vemurafenib and PLX8394 treatments; Supplemental Figure 5: PBRT characterization; Supplemental Figure 6: Targeting AKT and PDGFRβ in PBRT cell lines does not resensitize to PLX8394; Supplemental Figure 7: BRAF V600E splice variant analysis of PBRT cell lines and signaling and growth response of second-line therapies; Supplemental Methods.

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K–mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice variant–expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. Mol Cancer Ther; 17(1); 84–95. ©2017 AACR.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) drives several hallmark processes of cancer, making the enzymes that synthesize S1P, that is, sphingosine kinase 1 and 2 (SK1 and SK2), important molecular targets for cancer drug development. ABC294640 is a first-in-class SK2 small-molecule inhibitor that effectively inhibits cancer cell growth in vitro and in vivo. Given that AR and Myc are two of the most widely implicated oncogenes in prostate cancer, and that sphingolipids affect signaling by both proteins, the therapeutic potential for using ABC294640 in the treatment of prostate cancer was evaluated. This study demonstrates that ABC294640 abrogates signaling pathways requisite for prostate cancer growth and proliferation. Key findings validate that ABC294640 treatment of early-stage and advanced prostate cancer models downregulate Myc and AR expression and activity. This corresponds with significant inhibition of growth, proliferation, and cell-cycle progression. Finally, oral administration of ABC294640 was found to dramatically impede xenograft tumor growth. Together, these pre-clinical findings support the hypotheses that SK2 activity is required for prostate cancer function and that ABC294640 represents a new pharmacological agent for treatment of early stage and aggressive prostate cancer.Implications: Sphingosine kinase inhibition disrupts multiple oncogenic signaling pathways that are deregulated in prostate cancer. Mol Cancer Res; 13(12); 1591–601. ©2015 AACR.

Supplementary Figure Legends - PDF file 66K, Figure legends corresponding to supplemental figures

Contains Figure S1; SK2 siRNA validates Myc depletion by ABC294640. Supplemental methods for figure S1 also included.

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Supplementary Figure 7 - PDF file 79K, Supplemental data further demonstrating that androgen deprivation sensitizes CRPC cells to IR

Supplementary Table 1 - PDF file 282K, Supplemental table listing primer sequences utilized throughout study

Supplementary Figure 3,4 - PDF file 443K, Supplemental data demonstrating that exposure to IR promotes cell cycle arrest and AR activity regulates genes required for DNA damage repair

Supplementary figures

Supplemental Table 1: Patient data and treatment history Supplemental Table 2: PBRT #15 Ion Torrent results Supplemental Table 3: PBRT #16 Ion Torrent results

Supplementary Figures 1-6

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3′ untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. Cancer Res; 77(18); 5011–25. ©2017 AACR.

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.

HuR regulates all isoforms of PARG by binding a discrete minimal binding region (MBR) within its 3'UTR in both DDR- proficient MIA PaCa-2 and DDR- deficient Hs 766t cells. Such PARG mRNA and protein expression is also increased with other non-PARPi stress such as gemcitabine and oxaliplatin treatment, which simultaneously induce HuR cytoplasmic translocation.

LNCaP GSEA analyses RNAseq data.

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.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

LAPC4 phosphoproteomic data

Cbioportal analyses

Primer list

Figure legends corresponding with Supplemental Figure 1-6.

PARG rescue in HuR silenced cells restores PARPi resistance. Small-molecule inhibition of HuR prevents its cytoplasmic accumulation, abrogates PARG mRNA regulation and increases PARPi sensitivity, independent of DDR status.

LAPC4 aggressive phenotypes

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.

Doxycycline induction of HuR silencing in MIA PaCa-2 cells (sh290) downregulates PARG expression in vitro and tumor growth in vivo.

LAPC4 GSEA analyses RNAseq data

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.

HuR silencing results in downregulation of PARG expression, which prevents efficient removal of PAR polymers with or without PARPi stress. Expression and function of other PAR removing enzymes is not affected with HuR silencing.