Newman, Hannah

Abstract TET2 mutations (mTET2) are common genetic events in myeloid malignancies and clonal hematopoiesis. These mutations arise in the founding clone and are implicated in many clinical sequelae associated with oncogenic feedforward inflammatory circuits. However, the direct downstream effector of mTET2 responsible for the potentiation of these inflammatory circuits is unknown. To address this, we performed scRNA-seq and scATAC-seq in patients with COVID-19 with and without TET2-mutated clonal hematopoiesis reasoning that inflammation from COVID-19 may highlight critical downstream transcriptional targets of mTET2. Using this approach, we identified metastasis-associated lung adenoma transcript 1 (MALAT1), a therapeutically tractable lncRNA, as a central downstream effector of mTET2 that is both necessary and sufficient to induce the oncogenic proinflammatory features of mTET2 in vivo. We also elucidate the mechanism by which mTET2 upregulate MALAT1 and describe an interaction between MALAT1 and p65, which leads to RNA “shielding” from protein phosphatase 2A dephosphorylation, thus preventing resolution of inflammatory signaling. Significance: This work identifies MALAT1 as a requisite downstream effector of oncogenic feedforward inflammatory circuits necessary for the development of TET2-mutated CH and fulminant myeloid malignancy. We elucidate a novel mechanism by which MALAT1 “shields” p65 from dephosphorylation to potentiate this circuit and nominate MALAT1 inhibition as a future therapeutic strategy.

Abstract Purpose: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. Experimental Design: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. Results: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. Conclusions: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.

In this phase 1 trial, inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with clinically meaningful responses in 5 of 15 patients with relapsed or refractory chronic myelomonocytic leukemia (CMML). Preliminary data suggest that this approach may be tractable in CMML bearing activating NRAS mutations.

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.