Edmond Chipumuro

Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin.

The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding β-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143–54. ©2016 AACR.

Overcoming Resistance to the THZ Series of Covalent Transcriptional CDK Inhibitors

Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic strategy for cancers that rely on aberrant transcription; however, lack of understanding of resistance mechanisms to these agents will likely impede their clinical evolution. Here, we demonstrate upregulation of multidrug transporters ABCB1 and ABCG2 as a major mode of resistance to THZ1, a covalent inhibitor of CDKs 7, 12, and 13 in neuroblastoma and lung cancer. To counter this obstacle, we developed a CDK inhibitor, E9, that is not a substrate for ABC transporters, and by selecting for resistance, determined that it exerts its cytotoxic effects through covalent modification of cysteine 1039 of CDK12. These results highlight the importance of considering this common mode of resistance in the development of clinical analogs of THZ1, identify a covalent CDK12 inhibitor that is not susceptible to ABC transporter-mediated drug efflux, and demonstrate that target deconvolution can be accomplished through selection for resistance.

Abstract 2925: Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors

MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gene is challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC, a lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA) targeting MYC mRNA, is currently in Phase Ib/II clinical trials and showing promising results. In this study we used an improved EnCore LNP and MYC DsiRNA, which demonstrated MYC mRNA silencing activity and efficacy in mouse models of human hepatocellular carcinoma (HCC). Small molecule inhibitors that target BRD4; JQ1 and CDK7; THZ1 has previously reported anti-proliferative effects in various cancer types and efficacy in several tumor mouse models including HCC. Treatment with both JQ1 and THZ1 induced cell cycle arrest and apoptosis in various cancer cells by repressing MYC expression. Here we observed striking anti-proliferative effects in vitro when MYC-DsiRNA was combined with THZ1 in cancer cells. In addition, when mice bearing HCC tumors were treated with MYC-DsiRNA combined with either THZ1 or JQ1, the antitumor efficacy was additive or synergistic relative to either single agent alone. We observed significantly more MYC mRNA knockdown in the tumors that had the combination treatment compared to the tumors that received either of the single agent treatment. These preclinical data suggests the possibility of a significant and practical benefit of combining MYC-DsiRNA and small molecule inhibitors that target an epigenetic regulator BRD4 (JQ1) and a global gene regulator CDK7 (THZ1). Citation Format: Edmond Chipumuro, Zakir Siddiquee, Shanthi Ganesh, Serena Shui, Anee Shah, Boyoung Kim, Dongyu Chen, Purva Pandya, Rachel Storr, Weimin Wang, Hank Dudek, Cheng Lai, Marc Abrams, Bob Brown. Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2925.

Abstract 3827: Preclinical characterization of DCR-BCAT as a component of combination therapy

Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable of silencing any expressed mRNA with high specificity. DCR-MYC, a first-in-class DsiRNA targeting the MYC oncogene is currently in Phase 1b/II clinical trials [ASCO 2015, abstract 11006]. DCR-BCAT is an advanced preclinical development candidate that targets CTNNB1, the gene which encodes β-catenin. The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). Robust preclinical and genetic evidence strongly suggests that inhibiting β-catenin function would yield broad therapeutic benefit in oncology, but efforts to target it using conventional drug modalities have been unsuccessful to-date. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. Here, we explore DCR-BCAT as a monotherapy and in combination with both standard-of-care and experimental therapeutics. Interestingly, when mice bearing HCC tumors were treated with a combination of CTNNB1 and MYC DsiRNAs, the antitumor efficacy was additive or synergistic relative to either single agent alone. Additionally, since up to 50% of colorectal tumors have both activated Wnt signaling and mutant KRAS, we also explored combination therapy of DCR-BCAT and FDA-approved MEK inhibitors. A major advantage of DCR-BCAT is that the improved nanoparticle formulation is more selective for siRNA delivery to tumors over liver and other normal tissues. These data support continued development of DCR-BCAT as a first-in-class RNAi therapeutic, and highlight the potential for use in combination with other promising agents. Citation Format: Shanthi Ganesh, Wendy Cyr, Martin Koser, Girish Chopda, Edmond Chipumuro, Zakir Siddiquee, Kevin Craig, Serena Shui, Dongyu Chen, Cheng Lai, Hank Dudek, Weimin Wang, Bob Brown, Marc Abrams. Preclinical characterization of DCR-BCAT as a component of combination therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3827.

Abstract PR04: Targeting super-enhancer driven oncogene transcription through cyclin-dependent kinase inhibitors

Cyclin-dependent kinases (CDKs) with primary roles in transcription regulation are emerging as tractable therapeutic targets in cancers driven by the aberrant expression of oncogenic transcription factors. Our goal is to disrupt the myriad and pleomorphic features of oncogenic MYC through inhibiting CDKs involved in its transcriptional amplifier role. CDK7 participates in transcription initiation by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II and also functions as a CDK-activating kinase, while CDK12 functions in transcription elongation and RNA processing. Using a novel covalent CDK7 inhibitor, THZ1, we demonstrated striking activity and selectivity in neuroblastoma (NB) cells driven by high MYCN expression. This response translated to significant tumor regression in a mouse model of high-risk NB, without introducing discernible toxicity. We determined that this effect was associated with global inhibition of MYCN-dependent transcriptional amplification. THZ1 led to preferential downregulation of Pol II occupancy at super-enhancer-associated genes, including MYCN and other master transcription factors critical to neuronal development such as PHOX2B, GATA2, and DBH. Similarly, inhibition of CDK12 activity using a novel, first-in-class small molecule inhibitor, THZ-5-31-1, resulted in potent antitumor activity in MYCN-overexpressing NB cells. THZ-5-31-1 doses sufficient to induce PARP cleavage did not lead to significant inhibition of global transcription elongation. Rather, cytotoxicity was associated with preferential downregulation of RNA processing factors and a higher percentage of immature mRNA transcripts. Together, these results suggest that transcriptional CDK inhibitors, by affecting different aspects of the transcription machinery, may inhibit the growth of cancers driven by oncogenic transcription factors such as MYC. Citation Format: Malgorzata Krajewska, Nathan F. Moore, Edmond Chipumuro, Tinghu Zhang, Eugenio Marco, Clark Hatheway, Bandana Sharma, Nicholas Kwiatkowski, Guo-Cheng Yuan, Richard A. Young, Nathanael S. Gray, Rani E. George. Targeting super-enhancer driven oncogene transcription through cyclin-dependent kinase inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR04.

Abstract B74: Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma

Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALKF1174L, is sensitive to the FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would enhance the cytotoxic effect of crizotinib, we conducted a high throughput small molecule screen for compounds that synergize with crizotinib in NB cells expressing the ALKF1174L mutation. We identified two pan-selective cyclin dependent kinase (CDK) inhibitors, AT7519 and SNS-032, which have overlapping efficacy against the cell cycle-regulating CDK2 and transcriptional elongation-regulating CDK9. Both inhibitors demonstrated synergistic activity with crizotinib, leading to downregulation of pALK and downstream signaling and significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALKF1174L, but also in the other commonly observed ALKR1275Q mutation. Synergy was also noted with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. The combination of crizotinib and SNS-032 led to the inhibition of CDK9-mediated transcriptional elongation as indicated by downregulation of RNA polymerase II phosphorylation at serine 2. Additionally, the combination also induced proteolytic cleavage of elongation-regulating BRD4, suggesting alternative mechanisms contributing to transcriptional inhibition in addition to direct inhibition of CDK9 activity These findings were comparable with results obtained using compounds that were highly selective for CDK7, which has roles in transcriptional initiation as well as in CDK9 activation. Finally, in murine xenogaft models of ALK-mutated NB, the combination led to inhibition of tumor growth and prolongation of survival compared to single agents alone. Together, these data support further pre-clinical and clinical efforts to explore the therapeutic potential of combining ALK inhibitors with transcriptional CDK inhibitors in ALK-mutated NB. Citation Format: Nathan F. Moore, Edmond Chipumuro, Clark M. Hatheway, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B74.

Abstract PR05: Targeting of CDK7 inhibits super-enhancer-associated oncogenic programs in MYCN-amplified tumor cells

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a novel covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, would be useful therapy for cancers that are driven by MYC or its family members. Citation Format: Edmond Chipumuro, Eugenio Marco, Camilla L. Christensen, Nicholas Kwiatkowski, Tinghu Zhang, Clark M. Hatheway, Brian J. Abraham, Bandana Sharma, Caleb Yeung, Abigail Altabef, Antonio Perez-Atayde, Kwok-Kin Wong, Guo-Cheng Yuan, Nathanael S. Gray, Richard A. Young, Rani E. George. Targeting of CDK7 inhibits super-enhancer-associated oncogenic programs in MYCN-amplified tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR05.

Abstract 2195: A high-throughput chemical screen identifies synergistic activity between crizotinib and transcriptional CDK inhibitors in ALK-mutated neuroblastoma

Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALK F1174L , is sensitive to the FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would potentiate the effect of crizotinib, we performed a high-throughput compound screen using ALK F1174L -dependent human NB SH-SY5Y cells and compared viability between cells treated with the screen compound alone and in combination with crizotinib. The strongest “hits” among the ∼8,000 compounds screened were inhibitors of cyclin dependent kinases (CDKs). The combination of crizotinib and two candidate pan-selective CDK inhibitors, AT7519 and SNS-032 resulted in synergistic activity with significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALK F1174L , but also in those expressing ALK R1275Q , the most common NB-associated ALK mutation. Synergy was also seen with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. We determined that the synergistic effect was mediated preferentially through the transcriptional, rather than the cell cycle activity of these CDK inhibitors, denoted by absence of cell cycle arrest, stalling of RNA polymerase II at representative gene promoters, and loss of Pol II phosphorylation at the transcription elongation marker serine 2 in treated cells. These findings were comparable with results obtained using agents that were highly selective for CDK9 or CDK7, CDKs with roles in transcription regulation. Finally, in murine xenogaft models of ALK-mutated NB, the combination resulted in inhibition of tumor growth and prolongation of survival compared to single agents alone. Together, these data support further pre-clinical and clinical efforts to explore the therapeutic potential of combining ALK inhibitors with transcriptional CDK inhibitors in ALK-mutated NB. Citation Format: Nathan F. Moore, Edmond Chipumuro, Clark M. Hatheway, Tinghu Zhang, Nathanael S. Gray, Rani E. George. A high-throughput chemical screen identifies synergistic activity between crizotinib and transcriptional CDK inhibitors in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2195. doi:10.1158/1538-7445.AM2015-2195

Abstract LB-125: Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oncogenic MYC family transcription factors act as universal amplifiers of the existing gene expression program in many cancer cells, thus reducing rate-limiting constraints on growth and proliferation. Amplification of the MYCN gene defines approximately 50% of high risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. Here we exploit MYCN-driven global transcriptional amplification to specifically target MYCN-deregulated NB cells by inhibiting CDK7, a cyclin-dependent kinase with major roles in transcriptional initiation (as part of the TFIIH complex) and elongation (by activating CDK9/P-TEFb). For this purpose, we chose CDK7-IN-1, a newly developed, highly selective, first-in-class covalent inhibitor of CDK7, and then determined the effects of CDK7 inhibition on MYCN expression and global transcriptional activity. NB cells expressing high levels of MYCN were 10 times more sensitive to CDK7 inhibition than normal cells or NB cells not driven by amplified MYCN. CDK7-IN-1 was more active than its reversible (non-covalent) analogue and two pan-CDK inhibitors, roscovitine and flavopiridol. Cytotoxicity in treated MYCN -amplified NB cells resulted from G2 arrest and apoptosis. We observed a dose-dependent decrease in serine 2, 5 and 7 phosphorylation of RNA Pol II C-terminal domain only in MYCN -amplified NB cells, indicating that CDK7-IN-1 selectively inhibits RNA Pol II-mediated transcriptional initiation and elongation. Growth inhibition was accompanied by downregulation of MYCN and MYCN -associated transcriptional programs. CDK7-IN-1 significantly slowed tumor growth in a xenograft model of MYCN -amplified NB (median growth, 56.8% vs. 100% for vehicle-treated mice, P <0.05; n=6 per group) with tumors showing decreased MYCN expression. Mice remained free of toxicity over 4 weeks of CDK7-IN-1 treatment, suggesting that a therapeutic window may exist for NB cells with high MYCN expression. In conclusion, we show for the first time that selective suppression of MYCN expression and MYCN -associated transcriptional activity can be achieved through CDK7 inhibition, with associated antitumor effects in high-risk NB. Thus, CDK7 inhibition warrants further attention as a potential therapeutic strategy for MYCN-deregulated NB and perhaps other MYC-driven cancers. Citation Format: Edmond Chipumuro, Eugenio Marco, Tinghu Zhang, Camilla Christensen, Nicholas Kwiatkowski, Bandana Sharma, Clark Hatheway, Abigail Altabef, Brian J. Abraham, Kwok-Kin Wong, Guo-Cheng Yuan, Richard A. Young, Nathanael S. Gray, Rani E. George. Selective inhibition of CDK7 targets MYCN-driven transcriptional amplification in neuroblastoma. [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 LB-125. doi:10.1158/1538-7445.AM2014-LB-125