Supplementary Data from Transcriptional Plasticity Drives Leukemia Immune Escape

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth in vitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis in vivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.

Abstract Relapse of acute myeloid leukemia (AML) after allogeneic bone marrow transplantation has been linked to immune evasion due to reduced expression of major histocompatibility complex class II (MHCII) genes through unknown mechanisms. In this work, we developed CORENODE, a computational algorithm for genome-wide transcription network decomposition that identified a transcription factor (TF) tetrad consisting of IRF8, MYB, MEF2C, and MEIS1, regulating MHCII expression in AML cells. We show that reduced MHCII expression at relapse is transcriptionally driven by combinatorial changes in the expression of these TFs, where MYB and IRF8 play major opposing roles, acting independently of the IFNγ/CIITA pathway. Beyond the MHCII genes, MYB and IRF8 antagonistically regulate a broad genetic program responsible for cytokine signaling and T-cell stimulation that displays reduced expression at relapse. A small number of cells with altered TF abundance and silenced MHCII expression are present at the time of initial leukemia diagnosis, likely contributing to eventual relapse. Significance: Our findings point to an adaptive transcriptional mechanism of AML evolution after allogeneic transplantation whereby combinatorial fluctuations of TF expression under immune pressure result in the selection of cells with a silenced T-cell stimulation program. This article is highlighted in the In This Issue feature, p. 369

Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.

Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)-rearranged samples were also distinct from non-KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE- samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE- patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

Abstract Transcriptional deregulation is a hallmark of many cancers, including a subset that are “transcriptionally addicted” and depend on high levels of transcription for oncogenic program genes. Although these tumors are thought to be highly susceptible to targeting of the transcriptional apparatus, their molecular definition remains challenging. To better define molecular sensitivity to transcriptional inhibition, we profiled pan-cancer sensitivity to KB-0742 — a potent, selective, and orally bioavailable small molecule inhibitor of the transcription elongation cofactor CDK9. Multiplexed sensitivity profiling across ~1,000 adherent and suspension immortalized cell lines using the Broad PRISM platform revealed lineage and molecular determinants of sensitivity. Consistent with prior reports, MYC genomic amplification emerged as a key driver of CDK9 inhibitor sensitivity, and this was especially pronounced in non-small cell lung cancer. Sensitivity to CDK9 inhibition was further observed in MYC amplified/over-expressed ex vivo primary patient tumor cell cultures and patient-derived xenografts. Analysis of the temporal kinetics of CDK9 inhibition revealed a rapid collapse of oncogenic transcription programs comprised largely of short half-life transcripts including key oncogenes such as MYC and MCL1. In CDK9 sensitive ex vivo and in vivo models, suppression of oncogenic transcription for >8 hours was followed by apoptosis. In ex vivo models, CDK9 inhibitor sensitivity was observed for both treatment naïve and heavily pretreated patient samples. For in vivo models, CDK9 inhibition on an intermittent dosing schedule achieved sustained target coverage, as evidenced by both direct readouts of CDK9 activity and corresponding transcriptional response, and ultimately resulted in sustained tumor growth inhibition in multiple solid tumor types. These data suggest that MYC genomic amplification may serve as an important feature defining sensitivity to CDK9 inhibition in patients with advanced solid tumors. Citation Format: Melinda A. Day, Nikolaus D. Obholzer, Akanksha Pandey, Tom Chen, Tong Liang, Rosa A. Villagomez, Mulini Pingili, Jost V. Koren, David B. Freeman, Holly M. Nguyen, Jennifer L. Conor, Eva Corey, Matthew G. Reese, Andrew Boghossian, Brienne Engel, Melissa M. Ronan, Jennifer A. Roth, Joesph P. Vacca, Peter B. Rahl, Marius S. Pop, Benjamin W. Trotter, Charles Y. Lin, Jorge C. DiMartino, Pavan Kumar, Douglas C. Saffran. CDK9 inhibition is selective for transcriptionally addicted tumors harboring MYC genomic amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1141.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

Abstract Castration resistant prostate cancers (CRPCs) lose sensitivity to hormone therapy, but remain dependent on oncogenic transcription programs driven by the androgen receptor (AR) and other oncogenic transcription factors such as MYC. Using small molecule microarrays (SMMs), we screened HEK293 cellular lysates for compounds binding to exogenously expressed ARv7, a mutant splice form of AR that drives castration resistance. Although transcription factors like ARv7 and MYC are considered classically undruggable, SMMs are able to identify small molecule interactors of druggable co-factors and other proteins in complex with the target protein – in this case ARv7. SMM hits were triaged for the ability to selectively inhibit an AR dependent transcriptional reporter, and also for their ability to reduce proliferation in AR dependent tumor cells. From this screen, we identified KI-ARv3, a potent and selective inhibitor of CDK9. CDK9 is a cyclin-dependent kinase (CDK) that functions primarily as a general co-factor in RNA Polymerase II (RNA Pol II) transcription elongation. CDK9 is a well-characterized and important cofactor for AR, MYC, and other oncogenic transcription factors. In prostate cancer, CDK9 has been shown to modulate and be required for AR-specific gene expression. More broadly, transcriptional CDK inhibitors including those selective for CDK9 have shown strong potential as therapeutic agents owing to their ability to selectively downregulate oncogenic transcription programs and target tumors addicted to transcription factors such as AR or MYC. However, as CDK9 also plays a global role in transcription, it is unclear whether there exists a sufficient therapeutic index for clinical benefit. Prior clinical investigation of transcriptional CDK inhibitors has also been confounded by off-target interactions with other kinases and especially other CDKs that also play important roles in transcription and the cell cycle. We found that KI-ARv3 demonstrated excellent selectivity for CDK9 versus other CDKs and kinases, and further optimization of KI-ARv3 resulted in KB-00130742, an oral bioavailable CDK9 inhibitor with a biochemical IC50 of 15nM against CDK9 and greater than 50-fold selectivity for all profiled CDKs and greater than 100-fold selectivity against cell cycle CDKs. Both KI-ARv3 and KB-00130742 exhibited potent anti-tumor activity in CRPC models, as well as other models known to be dependent on MYC-driven transcription. In 22Rv1 CRPC cells, KB-00130742 rapidly downregulated nascent transcription, and preferentially depleted short half-life transcripts and AR driven oncogenic programs. In vivo, oral administration of KB-00130742 was well-tolerated and significantly reduced tumor growth in models of CRPC and leukemia. Overall these data support CDK9 inhibition using KB-00130742 as a therapeutic strategy to target AR dependence in CRPC and oncogenic transcription in other tumor types. Citation Format: André Richters, David Freeman, Christina Lee, Florian Kabinger, Shelby Doyle, Becky Leifer, Peter Mikochik, Sajjeev Jagannathan, Jost Vrabic Koren, Kristen Karlin, Calla M. Olson, Christopher Wilfong, Charles Y. Lin, Doug Saffran, Joseph Vacca, Norbert Bischofberger, Marius Pop, Angela N. Koehler. Targeting oncogenic transcription in prostate cancer with a novel, oral bioavailable, and ultra-selective CDK9 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1771.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.