Jing-Fang Lu

Combined Tumor Suppressor Defects Characterize Clinically Defined Aggressive Variant Prostate Cancers

Purpose: Morphologically heterogeneous prostate cancers that behave clinically like small-cell prostate cancers (SCPC) share their chemotherapy responsiveness. We asked whether these clinically defined, morphologically diverse, “aggressive variant prostate cancer (AVPC)” also share molecular features with SCPC. Experimental Design: Fifty-nine prostate cancer samples from 40 clinical trial participants meeting AVPC criteria, and 8 patient-tumor derived xenografts (PDX) from 6 of them, were stained for markers aberrantly expressed in SCPC. DNA from 36 and 8 PDX was analyzed by Oncoscan for copy number gains (CNG) and losses (CNL). We used the AVPC PDX to expand observations and referenced publicly available datasets to arrive at a candidate molecular signature for the AVPC. Results: Irrespective of morphology, Ki67 and Tp53 stained ≥10% cells in 80% and 41% of samples, respectively. RB1 stained <10% cells in 61% of samples and AR in 36%. MYC (surrogate for 8q) CNG and RB1 CNL showed in 54% of 44 samples each and PTEN CNL in 48%. All but 1 of 8 PDX bore Tp53 missense mutations. RB1 CNL was the strongest discriminator between unselected castration-resistant prostate cancer (CRPC) and the AVPC. Combined alterations in RB1, Tp53, and/or PTEN were more frequent in the AVPC than in unselected CRPC and in The Cancer Genome Atlas samples. Conclusions: Clinically defined AVPC share molecular features with SCPC and are characterized by combined alterations in RB1, Tp53, and/or PTEN. Clin Cancer Res; 22(6); 1520–30. ©2015 AACR.

Abstract 3808: Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Exportin 1/ Chromosomal Maintenance Protein 1 (XPO1/CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of tumor suppressor proteins such as p53, FOXO, PTEN, pRB and I-κB. Selinexor is an orally bioavailable XPO1 inhibitor that represents a novel class of small molecule compounds with potent activity against a wide variety of cancers. Here we report the activity of Selinexor against Castration resistant prostate cancer (CRPC). CRPC progression is mediated by activation of various adaptive Androgen Receptor (AR) signaling pathways. These pathways are currently being targeted by novel anti-androgen therapies such as abiraterone acetate and enzalutamide with favorable outcomes. However, resistance to anti-androgen therapy can be developed through deregulation of tumor suppressor pathways. XPO-1 is highly expressed in prostate cancer cells and therefore we tested the effects of Selinexor on CRPC cells and tumors models. Treatment of the C4-2B prostate cancer cell line with Selinexor in vitro significantly inhibited cell proliferation and resulted in nuclear accumulation of p53 and p21. In addition, Selinexor inhibited expression of the tumor-promoting gene Ubiquitin-Conjugating Enzyme E2C (UBE2C), which is activated by AR in these cells. Selinexor was also tested in tumor graft models of two patient tumors in castrated male mice. The MDA-PCa-133 tumor is an adenocarcinoma derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumors in castrated male mice express full-length AR and Prostate-Specific Antigen (PSA) but lack expression of p53. The MDA-PCa-144-13 tumor is a small cell carcinoma derived from a lethal variant of prostate cancer with anaplastic clinical phenotype and does not express AR or PSA but expresses a gain-of-function p53 mutant. Vehicle or Selinexor treatment (10 mg/kg p.o. QoDX3 M/W/F) of MDA-PCa-133 tumors for 34 days resulted in almost complete inhibition of tumor growth with a 30-fold reduction of PSA (p 8 fold compared to vehicle only; p<0.0047). Immunohistochemical analysis of MDA-PCa-133 tumors showed that Selinexor induced nuclear accumulation of XPO1 cargos FOXO3a and p27 with concomitant reduction in the proliferation marker KI67. In conclusion, Selinexor demonstrated robust inhibition of CRPC tumor growth and activated multiple tumor suppressors in prostate cancer cells. Selinexor is now being evaluated in Phase 1 clinical studies in patients with advanced hematological and solid tumor cancers, and preliminary results show adequate tolerability with evidence of anti cancer activity. Future clinical studies in patients with CRPC are planned and will provide more information on the use of Selinexor as monotherapy and in combination with anti-androgen therapy of CRPC. Citation Format: Sankar Narayan Maity, Guanglin Wu, Jing-Fang Lu, Anh Hoang, Yosef Landesman, Dilara McCauley, Sharon Shacham, Michael G. Kauffman, Ana M. Aparicio, Eleni Efstathiou, John C. Araujo, Christopher J. Logothetis. Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2014-3808

Abstract 2986: Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma.

Small cell prostate cancer (SCPC) is an androgen receptor (AR) negative variant that often emerges in the castration-resistant progression of typical AR-positive prostate adenocarcinomas. Its presence predicts for an aggressive clinical course with poor response to hormonal therapies and a short median survival. Evidence suggests that the emergence of (SCPC) occurs through cellular transdifferentiation. Therefore, we hypothesized that loss of AR gene transcription may depend on epigenetic events. Given the central role of AR in prostate cancer, we reasoned that identifying the mechanisms involved in its downregulation could improve our understanding of how the lethal SCPC subtype arises. Here we investigated the DNA methylation and histone modifications of AR in a group of six prostate tumor xenografts developed from men with CRPC (two AR-positive [MDA PCa 170.4 and MDA PCa 180.30] and four AR-negative SCPC [MDA PCa 144.13, MDA PCa 144.4, MDA PCa 155 and MDA PCa 146.10]) and three established prostate cancer cell lines (one AR-positive [LNCaP] and two AR-negative [PC3 and DU145]). We first evaluated the methylation status of selected CpG sites in the AR promoter-associated CpG island using bisulfite-sequencing followed by pyrosequencing analysis. Except for the cancer cell lines PC-3 and DU145, all other cell lines and xenografts were unmethylated regardless of the AR expression status. Next, we used chromatin immunoprecipitation (ChIP) to evaluate marking of the AR promoter and a putative proximal enhancer by active (H3K4me3 and H3K9ac) and repressive (H3K9me2 and H3K27me3) histone modifications. Marking of the constitutively expressed genes (ACTB and GAPDH) and a repressed gene (HBB) served as control for these experiments. As expected, the only samples with AR marking by H3K4me3 and H3K9ac were the two AR-positive xenografts and LNCaP. Marking by repressive histone modifications was more variable: H3K27me3 was a universal finding in AR-negative samples except for Du-145, and was accompanied by H3K9me2 in two out of four xenografts. H3K27me3 and H3K9me2 also marked the putative enhancer region when AR was repressed. Altogether, these data indicate that promoter DNA methylation of AR is an infrequent finding in prostate cancer, while polycomb protein-based silencing is nearly universal in AR-negative variants. Also, it shows that H3K9me2 promotes reinforcement of silencing. Thus, we propose that reactivation of AR may by achieved in selected cases by inhibiting H3K27me3 alone, while concomitant inhibition of both polycomb and SET domain proteins may be necessary for double-positive H3K27/K9 methylation cases. Given the purported differentiating and tumor suppressor effects of AR, its reactivation in SCPC may be of therapeutic benefit. Citation Format: Brittany N. Kleb, Marcos RH Estecio, Guanglin Wu, Jing-Fang Lu, Christopher J. Logothetis, Sankar Maity, Ana M. Aparicio. Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2013-2986

Abstract 4772: Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cytochrome P450 17α-hydroxylase /17,20-lyase (CYP17) is a validated treatment target for metastatic castrate-resistant prostate cancer (mCRPC). Abiraterone acetate (AA; Zytiga) inhibits 17α-hydroxylase and 17,20-lyase reactions catalyzed by CYP17 and thus reduces androgen biosynthesis. However, co-administration of prednisone is required to suppress the mineralocorticoid excess from 17α-hydroxylase inhibitions. VT-464, a non-steroidal small molecule inhibits androgen production without mineralocorticoid excess or cortisol depletion by selective inhibition of CYP17 17,20-lyase. We determined the impact of VT-464 on tumor growth of a mCRPC xenograft, MDA-PCa-133, in vivo, and on androgen signaling in C4-2B prostate cancer cells in vitro. The MDA-PCa-133 xenograft is derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumor expresses PSA, full-length androgen receptor (AR) and AR-V7 isoform. We determined the effect of VT464 and AA on MDA-PCa-133 growing in tumor-bearing castrated male mice: randomization into three groups; oral treatment with vehicle only, VT-464, (100 mg/kg bid), or AA (100 mg/kg bid) for 25 days. Both VT-464 and AA reduced tumor volume (>two fold compared to vehicle; p<0.05). These results indicate that selective VT-464 CYP17 lyase inhibition is as effective as AA CYP17 inhibition in this model. We determined the effects of VT-464 on AR-dependent luciferase reporter activity and expression of AR signaling genes, PSA and NKX3.1, in cultured C4-2B prostate cancer cells with or without VT-464 or AA treatment. VT-464 or AA highly reduced AR-dependent reporter activity and specific expression of PSA and NKX3.1. AR-dependent signaling in the presence of VT-464 or AA was restored however by 10 nM DHT, consistent with VT-464- or AA-mediated depletion of androgen in C4-2B cells. The AR-dependent reporter activity was also measured following overexpression of full-length AR or AR-V7 isoform in C4-2B cells. Full-length AR-dependent reporter activity was (>80%) inhibited; AR-V7-dependent reporter activity was inhibited by 50% by 1 μM VT-464 or AA. We conclude that VT-464 inhibited AR signaling as effectively as AA in C4-2B prostate cancer cells. The findings suggest that AR signaling in cells, which express high levels of AR isoform (AR-V7) are more resistant to androgen biosynthesis inhibitors than those with wild type AR. Preliminary observations suggest C4-2B cells with AR-V7 are less sensitive to AA than VT- 464. Ongoing clinical and co-clinical studies will determine if the observed differences in steroid profile and relative efficacy of VT-464 compared to AA are clinically and biologically meaningful. Citation Format: Sankar N. Maity, Guanglin Wu, Jing-fang Lu, Joel R. Eisner, Stephen W. Rafferty, Robert J. Schotzinger, William R. Moore, Christopher J. Logothetis, John C. Araujo, Eleni Efstathiou. Efficacy of VT-464, a novel selective inhibitor of cytochrome P450 17,20-lyase, in castrate-resistant prostate cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2013-4772

Abstract 344: Targeting fibroblast growth factor signaling in prostate cancer

Currently no therapy effectively controls the progression of advanced human prostate cancer. We previously reported that fibroblast growth factor 9 (FGF9) contributes to the osteoblastic progression of human prostate cancer in bone (J Clin Invest 2008;118:2697). We examined the effect of TKI258 (a receptor tyrosine kinase inhibitor [TKI] with strong activity against FGF receptor 1-3 [FGFR1-3; IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 344.

Abstract C253: Effective inhibition of Hedgehog signaling pathway with small molecule Smoothened inhibitor GDC‐0449 in a bone producing prostate cancer xenograft

Background: The tumor microenvironment is involved in prostate cancer progression. Several stromal‐epithelial‐interacting pathways associated with prostate cancer (PCa) progression in the bone, are also considered candidates of resistance to current therapeutic strategies applied. Hedgehog (Hh) signaling has been associated with PCa aggressiveness. Therefore Hh pathway could be a novel diagnostic and therapeutic target, also contributing to therapy against prostate carcinoma metastasis in bone. Experimental work on specific chemical inhibitors of Hh signaling suggests that they may represent an entirely new class of therapeutic agents. In this study we tested the efficacy of Hh pathway inhibition with Smoothened inhibitor GDC‐0449 in 118b prostate cancer xenograft model. Experimental Design: We employed the xenograft MDA PCa 118b, derived from the bone metastasis of a patient with castrate‐resistant prostatic adenocarcinoma. This is characterized by unique bone producing feature when injected subcutaneously in SCID mice. Bone is the most common site for metastasis of prostate carcinoma, therefore a model with the new bone formation distinctive characteristic is really informative. Following MDA PCa 118b sc injection, animals were treated with Smoothened inhibitor GDC‐0449 (oral administration, for 21 days). Hh pathway components expression was measured both in the tumor cells and stromal compartment using species specific primers for real‐time PCR, western blot analysis and immunohistochemistry. Results: Paracrine Hedgehog signaling was confirmed in the tumor xenograft microenvironment. Baseline Shh expression was present in human tumor cells and significantly higher compared to stromal compartment, while nuclear Gli1 (a hallmark of pathway activation) and Ptch1 expression in the stromal compartment were present and significantly higher compared to human tumor cells. Smoothened inhibition resulted in attenuation of stromal expression of Gli1 and Ptch1, in the treated group compared to the control thus confirming the pharmacodynamic effect of the agent. A trend for slower tumor growth at the treated group was observed, while a decline in proliferation rate was noted when Hh pathway was suppressed in the stromal compartment. Conclusion: Hh signaling pathway demonstrates paracrine activation in the 118b prostate cancer bone producing xenograft model. GDC‐0449 is effective in blocking Hh signaling in PCa. Future directions: A combination of Hedgehog signaling inhibition and cytotoxic therapies, such as androgen ablation would likely increase efficacy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C253.