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Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.Significance:Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Video Legends and Supplementary Figures 1-10

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Supplementary Table from Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML

Supplementary Figure from Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation

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Cell lines used in this study.

Supplementary Figure from Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML

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Supplementary Methods, Supplementary References, Supplementary Figure 1 - in vitro analysis of various BET inhibitors against Eu-myc lymphoma cells and effect of JQ1 on OPM2 and OPM2/BCL2 cells; Supplementary Figure 2 - In vitro effects of JQ1 on OPM2 and OPM2/BCL2 cells; Supplementary Figure 3 - In vivo effects of JQ1 showing lack of toxicity; Supplementary Figure 4 - induction of Bim in JQ1-sensitive and JQ1-resistant Eu-myc lymphomas; Supplementary Figure 5 - Activated Ras confers resistance to JAK inhibitor and JQ1.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.

SummaryMost human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function-first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

Background Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model. Results We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation. Conclusions These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases. Graphical Abstract

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias. Loss of function of the minor spliceosome component ZRSR2 enhances hematopoietic stem cell self-renewal through minor intron retention of its target LZTR1, which is a regulator of RAS-related GTPases. Minor intron retention of LZTR1 was also identified in Noonan syndrome and diverse solid tumor types.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments.

BACKGROUND: Covalent (irreversible) Bruton’s tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell–receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)

Dysregulation of the epigenome drives aberrant transcriptional programmes that promote cancer onset and progression. Although defective gene regulation often affects oncogenic and tumour-suppressor networks, tumour immunogenicity and immune cells involved in antitumour responses may also be affected by epigenomic alterations. This could have important implications for the development and application of both epigenetic therapies and cancer immunotherapies, and combinations thereof. Here, we review the role of key aberrant epigenetic processes - DNA methylation and post-translational modification of histones - in tumour immunogenicity, as well as the effects of epigenetic modulation on antitumour immune cell function. We emphasize opportunities for small-molecule inhibitors of epigenetic regulators to enhance antitumour immune responses, and discuss the challenges of exploiting the complex interplay between cancer epigenetics and cancer immunology to develop treatment regimens combining epigenetic therapies with immunotherapies.

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation. Significance: Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221

Peripheral T-cell lymphoma (PTCL) is a rare, heterogenous malignancy with dismal outcomes at relapse. Hypomethylating agents (HMA) have an emerging role in PTCL, supported by shared mutations with myelodysplasia (MDS). Response rates to azacitidine in PTCL of follicular helper cell origin are promising. Guadecitabine is a decitabine analogue with efficacy in MDS. In this phase II, single-arm trial, PTCL patients received guadecitabine on days 1–5 of 28-day cycles. Primary end points were overall response rate (ORR) and safety. Translational sub-studies included cell free plasma DNA sequencing and functional genomic screening using an epigenetically-targeted CRISPR/Cas9 library to identify response predictors. Among 20 predominantly relapsed/refractory patients, the ORR was 40% (10% complete responses). Most frequent grade 3-4 adverse events were neutropenia and thrombocytopenia. At 10 months median follow-up, median progression free survival (PFS) and overall survival (OS) were 2.9 and 10.4 months respectively. RHOAG17V mutations associated with improved PFS (median 5.47 vs. 1.35 months; Wilcoxon p = 0.02, Log-Rank p = 0.06). 4/7 patients with TP53 variants responded. Deletion of the histone methyltransferase SETD2 sensitised to HMA but TET2 deletion did not. Guadecitabine conveyed an acceptable ORR and toxicity profile; decitabine analogues may provide a backbone for future combinatorial regimens co-targeting histone methyltransferases.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance anti-tumor immunity and augment cancer immunotherapies. By screening a small molecule library of epigenetics-based therapeutics, BET bromodomain inhibitors (BETi) were identified as agents that promote the anti-tumor activity of CD8+ T-cells. BETi sensitized diverse tumor types to the cytotoxic effects of the pro-inflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of pro-survival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific (TCB) antibodies increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune oncology agents.STATEMENT OF SIGNIFICANCEManipulating the epigenome is an evolving strategy to enhance anti-tumor immunity. We demonstrate that BET bromodomain inhibitors potently sensitize solid tumors to CD8+ T-cell killing in a TNF-dependent manner. This immunomodulatory mechanism can be therapeutically leveraged to augment immuno-oncology therapies, including TCB antibodies and immune checkpoint blockade.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Pharmacological inhibition of epigenetic enzymes can have therapeutic benefit, particularly against hematological malignancies. While these agents can affect tumor cell growth and proliferation, recent studies have demonstrated that pharmacological de-regulation of epigenetic modifiers may additionally mediate anti-tumor immune responses. Here we discovered a novel mechanism of immune regulation through the inhibition of histone deacetylases (HDACs). In a genetically engineered model of t(8;21) AML, leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor panobinostat required activation of the type I interferon (IFN) signaling pathway. Plasmacytoid dendritic cells (pDCs) were identified as the cells producing type I IFN in response to panobinostat, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated activation of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, while combined treatment of panobinostat and recombinant IFNα improved therapeutic outcomes. These discoveries offer a new therapeutic approach for t(8;21) AML and demonstrate that epigenetic rewiring of pDCs enhances anti-tumor immunity, opening the possibility of exploiting this cell type as a new target for immunotherapy.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. Significance: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397

SUMMARYGene expression is tightly controlled by Cyclin-dependent kinases (CDKs) which regulate the RNA Polymerase II (RNAPII) transcription cycle at discrete checkpoints. RNAPII pausing is a CDK9-controlled rate-limiting process that occurs shortly after initiation and is required for spatio-temporal control of transcription in multicellular organisms. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII, and is recruited to transcription pausing sites by INTS6, a subunit of the Integrator complex. INTS6 depletion disrupts the Integrator-PP2A association and confers resistance to CDK9 inhibition. This results in unrestrained activity of CDK9 and dysregulation of acute transcriptional responses. Pharmacological PP2A activation amplifies RNAPII pausing mediated by CDK9 inhibitors and synergizes therapeutically in a model of MLL-rearranged leukemia. These data demonstrate that finely-tuned gene expression relies on the delicate balance of kinase and phosphatase activity throughout the transcription cycle.HIGHLIGHTSLoss of INTS6 confers resistance to CDK9 inhibitionINTS6 recruits PP2A to Integrator and chromatinPP2A/INTS6 complexes functionally oppose CDK9PP2A/INTS6 fine-tune acute transcriptional responsesSynergistic anti-cancer activity between PP2A activators and CDK9 inhibitors

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD–driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD–induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. Significance: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD–driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD–driven AMLs to chemotherapy. This article is highlighted in the In This Issue feature, p. 1307

CDK12 and CDK13 regulate POLII elongation rate and processivity and influence the selection of transcription termination sites., The RNA polymerase II (POLII)–driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3′ polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.

To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.

Summary: JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite’s digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT’s native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials., Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediates multidrug resistance, but its natural function remains unclear. Here, Shafik et al. show that PfCRT transports host-derived peptides of 4-11 residues but not other ions or metabolites, and that drug-resistance-conferring PfCRT mutants have reduced peptide transport.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by two molecular subtypes, of which the basal-like subtype is associated with the worst survival and is highly resistant to first-line chemotherapy. We previously reported that keratin 17 (K17), a component of the molecular signature of the basal-like subtype, is a negative prognostic and predictive biomarker, whose overexpression results in chemoresistance and shortened patient survival. Here, we aimed to uncover the mechanistic role of K17 in driving cancer aggression and explore the therapeutic opportunities in K17-expressing PDAC. Beyond its role as a cytoskeletal protein, K17 can solubilize from the filamentous form and translocate into the nucleus, where it impacts multiple cellular properties. Previous reports suggest that nuclear K17 regulates cell-cycle progression and gene expression. However, the detailed mechanisms through which K17 modulates gene expression remains unexplored. Through domain-prediction analyses, we identified a novel chromatin-remodeling (SANT) domain in K17 that may interact with histones to regulate chromatin structure. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) revealed that K17 positive cells had comparably accessible regions relative to K17 negative cells. However, the deletion of the K17 SANT domain (dSANT) decreased chromatin accessibility, suggesting that nuclear K17 impacts chromatin structure via the SANT domain. With RNA sequencing (RNA-seq) pathway enrichment analysis, we found that K17 positive cells had differential expressed genes that are associated with cell signaling, immune regulation, and metabolic pathways. More specifically, by comparing wild type K17 and dSANT K17-expressing cells, we found that the SANT domain impacts a wide variety of immune regulatory pathways, many of which affect the tumor microenvironment. We are now performing chromatin immunoprecipitation sequencing (ChIP-seq) with acetylation of lysine 27 on histone H3 protein subunit (H3K27ac), to reveal differential bindings that are impacted by K17 and the SANT domain. Importantly, mice bearing orthotopic xenografts of PDACs with K17-SANT deletion showed survival comparable to those with K17 negative tumors and had significantly longer survival than those with wild-type K17. In summary, we identified a novel chromatin remodeling domain in K17 that could be explored as a target for the development of a biomarker-based treatment for PDAC. Citation Format: Chun-Hao Pan, Robert Tseng, Simon J. Hogg, Gabriella Baraks, Cindy V. Leiton, Lucia Roa-Peña, Natalia Marchenko, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. A novel chromatin remodeling domain of keratin 17 regulates transcription and promotes tumor aggression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-048.

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Mutations in RNA splicing factors are amongst the most common genetic alterations in myeloid malignancies. Mutations in the splicing factors SF3B1, SRSF2, and U2AF1 occur as heterozygous, missense mutations and have been shown to confer a change-of-function. In contrast, the X chromosome encoded ZRSR2 is enriched in nonsense/frameshift mutations in males, consistent with loss of function. To date however, we do not understand the basis for enrichment of ZRSR2 mutations in leukemia. Moreover, ZRSR2 is the only one of these factors that primarily functions in the minor spliceosome. While most introns are spliced by the major spliceosome, a small subset ( The rarity and conservation of minor introns offered a unique opportunity to investigate splicing factor mutations and identify potential tissue-specific roles of the minor spliceosome. Modeling loss-of-function mutations in ZRSR2 via a mouse model for induced deletion of Zrsr2 revealed strikingly enhanced self-renewal of Zrsr2-deficient male and female hematopoietic cells (Fig. A-C). This was in stark contrast to the effects of hotspot mutations in Sf3b1and Srsf2 and similar to those of Tet2 loss on increasing self-renewal and numbers of HSCs. Zrsr2 loss was also associated increased myeloid cells in the blood and long-term hematopoietic stem cells (HSCs) in the marrow (Fig. C). To understand the mechanistic basis by which ZRSR2 loss causes aberrant HSC self-renewal, we quantified transcriptome-wide splicing patterns in MDS patients. ZRSR2-mutant samples had widespread, dysfunctional recognition of minor introns- 48% of minor introns exhibiting significantly increased retention (Fig. D). We next systematically mimicked the effects of nonsense-mediated decay caused by minor intron retention in ZRSR2-mutants. Every gene containing a ZRSR2-regulated minor intron was targeted by 4 sgRNAs via a positive-enrichment CRISPR screen using pools of lentiviral sgRNAs in cytokine-dependent human and mouse hematopoietic cell lines. This identified several minor intron-containing genes whose downregulation conferred cytokine independence. Strikingly, just one gene was enriched in all lines (Fig. E): LZTR1, a cullin-3 adaptor for ubiquitin-mediated suppression of RAS-related GTPases which is subject to loss-of-function mutations in several cancers and the RASopathy Noonan Syndrome. Minor intron retention in LZTR1 correlated with reduced LZTR1 protein in MDS patients (Fig. F-G). Inducing mutations in either the protein-coding region of LZTR1 or its minor intron resulted in cytokine independence (Fig. H), reduced LZTR1, and dramatic accumulation of RIT1, a RAS GTPase substrate of LZTR1. In a Noonan Syndrome family wherein one child died of AML, the mother and all children carried an intronic mutation within LZTR1's minor intron (Fig. I-J). Fibroblasts from each family member revealed clear LZTR1 minor intron retention with impaired LZTR1 protein expression and RIT1 accumulation in subjects bearing the LZTR1 minor intron mutation (Fig. J). We next interrogated LZTR1 minor intron splicing across all cancers in the TCGA. While LZTR1's minor intron was efficiently excised in normal samples, a notable subset of tumors in almost all cancer types exhibited significantly increased retention within LZTR1's minor intron. These data indicate LZTR1 is frequently dysregulated via perturbed minor intron splicing - much more so than by protein-coding mutations alone. Here we uncover a heretofore unrecognized role of minor intron excision in regulating HSC self-renewal, a molecular link between ZRSR2 mutations and aberrant LZTR1 splicing and expression, and frequent LZTR1 minor intron retention in diverse cancers and cancer predisposition syndromes. Given frequent post-transcriptional disruption of LZTR1 in the absence of protein-coding mutations, our data additionally motivate study of other cancer-associated minor intron-containing genes which may be dysregulated via similar, and as-yet-undetected, aberrant splicing. Figure Disclosures Abdel-Wahab: Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy.

Summary BET inhibitors (BETi) target bromodomain-containing proteins and are currently being evaluated as anti-cancer agents. We find that maximal therapeutic effects of BETi in a Myc-driven B cell lymphoma model required an intact host immune system. Genome-wide analysis of the BETi-induced transcriptional response identified the immune checkpoint ligand Cd274 (Pd-l1) as a Myc-independent, BETi target-gene. BETi directly repressed constitutively expressed and interferon-gamma (IFN-γ) induced CD274 expression across different human and mouse tumor cell lines and primary patient samples. Mechanistically, BETi decreased Brd4 occupancy at the Cd274 locus without any change in Myc occupancy, resulting in transcriptional pausing and rapid loss of Cd274 mRNA production. Finally, targeted inhibition of the PD-1/PD-L1 axis by combining anti-PD-1 antibodies and the BETi JQ1 caused synergistic responses in mice bearing Myc-driven lymphomas. Our data uncover an interaction between BETi and the PD-1/PD-L1 immune-checkpoint and provide mechanistic insight into the transcriptional regulation of CD274., Graphical Abstract, Highlights • BETi require an intact host immune system to promote robust anti-tumor responses • BRD4 inhibition inhibits PD-L1 transcription independently from MYC expression • BRD4 and IRF1 co-regulate interferon-induced PD-L1 transcription • Combinations of BET inhibitor and immune modulating therapy are efficacious in vivo, Hogg et al. find that BET bromodomain inhibitors promote anti-tumor immune responses through transcriptional repression of immune checkpoint ligand PD-L1 in genetically diverse tumor models and in response to inflammatory stimuli. Moreover, BET inhibitors enhance the efficacy of immune modulating therapies, such as checkpoint blockade.

A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of ‘on-target’ activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.