Background Signal transducer and activator of transcription (STAT) activation of gene expression ... more Background Signal transducer and activator of transcription (STAT) activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1). Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFN)γ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II) occupancy at the IRF1 promoter....
Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to... more Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular prolifera...
The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the ... more The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results ...
Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic... more 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 accom...
Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent importan... more 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 ob...
Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable o... more 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-...
The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancer... more 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 ...
Cyclin-dependent kinases (CDKs) with primary roles in transcription regulation are emerging as tr... more 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 amplifica...
Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent importan... more 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 potentiate the effect of crizotinib, we performed a high-throughput compound screen using ALKF1174L-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 ALKF...
MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gen... more 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 indu...
Background Signal transducer and activator of transcription (STAT) activation of gene expression ... more Background Signal transducer and activator of transcription (STAT) activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1). Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFN)γ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II) occupancy at the IRF1 promoter....
Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to... more Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular prolifera...
The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the ... more The cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and appears to selectively affect the expression of DNA damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results ...
Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic... more 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 accom...
Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent importan... more 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 ob...
Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable o... more 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-...
The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancer... more 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 ...
Cyclin-dependent kinases (CDKs) with primary roles in transcription regulation are emerging as tr... more 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 amplifica...
Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent importan... more 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 potentiate the effect of crizotinib, we performed a high-throughput compound screen using ALKF1174L-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 ALKF...
MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gen... more 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 indu...
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Papers by Edmond Chipumuro