Myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit constitutive activity of JAK-STAT signaling due to mutations in JAK2, c-MPL or calreticulin genes. Additional mutations in chromatin/transcriptional modifiers (epimutations) induce transformation to AML (sAML) in up to 20% of patients with MPN-MF. Lack of significant activity of the JAK1 & 2 inhibitor (JAKi) ruxolitinib and of AML chemotherapy highlights the need to develop and test novel agents and combinations that would improve clinical outcome in patients with post- MPN sAML. Genetic alterations and dysregulated epigenome produce the dysregulated transcriptome responsible for the transformed phenotype and therapy-refractoriness in post-MPN sAML blast progenitor cells (BPCs). This dysregulated transcriptome is dependent on ?chromatin-reader? BET (bromodomain and extra-terminal) proteins (BETPs), e.g., BRD4, and on its interactor pTEFb (positive transcription elongation factor b), both recruited to super-enhancers and promoters of actively-transcribed oncogenes. Cyclin dependent kinase 9 (CDK9), the catalytic subunit of pTEFb, phosphorylates RNA pol II (RNAP2), promoting RNAP2-mediated mRNA transcript elongation of oncogenes essential for growth and survival of post-MPN sAML BPCs. However, effects of BETP-CDK9 axis inhibition on active super-enhancers/enhancers and promoters with resulting impact on the dysregulated transcriptome and survival have not been elucidated in patient-derived (PD) sAML BPCs. Additionally, epigenetic mechanisms of resistance to CDK9 or BETP inhibitor (CDK9i or BETi) treatment and their therapeutic abrogation in post-MPN sAML BPCs need evaluation. Our preliminary studies demonstrate that CDK9i or BETP-antagonist (BETi and BETP- PROTACs) treatment induces apoptosis of post-MPN sAML BPCs, which is associated with repression of sAML-relevant oncogenes, e.g., c-MYC, STAT3/5, NFkB, Bcl-xL and MCL-1. We hypothesize that BETP- antagonist and CDK9i-based combinations will repress the dysregulated transcriptome and oncogenes, and with JAKi or BCL2/Bcl-xL inhibitor co-treatment, synergistically induce in vitro and in vivo lethality in PD, post- MPN sAML BPCs.
Specific aims of these studies are:
Aim 1 : To elucidate the effects of BETP-PROTAC and CDK9i on active super-enhancers/enhancers (by ATAC-Seq and ChIP-Seq), mRNA transcriptome (by RNA-Seq) and on protein expressions (by CyTOF), as well as determine their pre-clinical efficacy against genetically-profiled, cultured cell lines and PD, post-MPN sAML BPCs.
Aim 2 : To determine lethal activity of BETP-PROTAC and CDK9i-based combinations against JAKi-sensitive and JAKi-persister/resistant sAML BPCs, utilizing in vitro cell cultures and in vivo xenograft models.
Aim 3 : To elucidate the dysregulated epigenome and transcriptome as well as susceptibility to BETP-PROTAC-based combinations in BETi- or CDK9i-persister/resistant post-MPN sAML BPCs.

Public Health Relevance

Studies proposed will determine the dysregulated epigenome and transcriptome, as well as lethal activity of BET protein degrader- and CDK9 inhibitor-based combinations, in patient-derived, genomically-profiled, post- myeloproliferative neoplasms (MPN) secondary (s) AML blast progenitor cells (BPCs). Additionally, these studies will determine in vivo activity of the BETP-PROTAC-based combinations against JAK-kinase inhibitor-, BET inhibitor- and CDK9 inhibitor-persister/resistant post-MPN sAML BPCs.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Developmental Therapeutics Study Section (DT)
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Venkatachalam, Sundaresan
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University of Texas MD Anderson Cancer Center
Internal Medicine/Medicine
United States
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