ALBERT

Description: Background: Activating mutations of NRAS and KRAS genes are common in newly diagnosed acute myeloid leukemia (AML), occurring in 11-16% and 4-5% of patients, respectively. RAS mutations are frequently acquired at time of progression from MDS to AML and are associated with poor survival. Next generation sequencing (NGS) at diagnosis and during complete remission has shown that RAS mutations have high clearance rates with induction chemotherapy. In the CALGB 8525 study, RAS-mutant younger patients (age

Description: Background: Chromosomal rearrangements involving the 11q23 locus, resulting in fusions of the Mixed Lineage Leukemia (MLL) gene, are found in 5-10% of adult patients with acute myeloid leukemia and can represent a poor prognostic feature. Patients with this subtype of AML often respond well to standard induction chemotherapy but frequently relapse, even after allogeneic hematopoietic stem cell transplantation. Effective therapy options in the relapsed / refractory setting for this high-risk group represent an urgent unmet clinical need. In MLL-rearranged AML, recruitment of disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine 79 (H3K79) methyltransferase, and subsequent changes in methylation of downstream targets HOXA9 and Meis1 is central to leukemogenesis. DOT1L-mediated expression of MLL target genes is critical to developing leukemia in a murine model and inhibition of DOT1L suppressed downstream expression of MLL target genes. Pinometostat is a potent and selective small molecule inhibitor of DOT1L methyltransferase activity. Use of pinometostat in a continuous IV infusion in a rat xenograft model of MLL-rearranged leukemia showed complete, sustained tumor regressions without significant appreciable toxicities. A recent single-agent, phase I trial evaluated pinometostat in R/R patients with MLL-R myeloid malignancies. This study included 43 patients with MLL-R AML and observed that the drug was generally well-tolerated. Responses to pinometostat included one patient with morphologic CR, one with cytogenetic CR, three with resolution of leukemia cutis, and nine with signs of differentiation / leukocytosis. Promoter hypermethylation contributes to the dysregulation of MLL-R target genes HOXA9 and Meis1, and this effect was reversed upon treatment with the hypomethylating agent azacitidine. As such, this combination may lead to a more robust response in this patient population. We now seek to combine the novel, targeted agent pinometostat with azacitidine in R/R MLL-R AML. Study design & methods: We are conducting an open-label, single-arm, phase Ib / II study that will enroll 36-48 patients with R/R MLL-R AML to evaluate the tolerability and preliminary efficacy of pinometostat in combination with azacitidine. Since MLL-R AML is a rare disease and is seen in only 5-10% of patients with AML, we are looking to collaborate with other centers through the National Cancer Institute Experimental Therapeutics Clinical Trials Network (ETCTN) to meet our accrual target. The study is active nationally and enrolling patients (NCT03701295). The presence of the MLL-R will be confirmed locally by FISH or cytogenetics. Adult patients who are refractory to two courses of induction, relapse after CR, or elect not to pursue induction therapy are considered eligible. Patients who have previously undergone HSCT or who have well-controlled HIV are also candidates. Pinometostat will be given by continuous IV infusion via portable pump. Azacitidine will be administered at 75mg/m2 daily over 7 days at the start of each 28-day cycle. Patients will be followed on trial for a total of 6 cycles, with bone marrow biopsies done for assessment after cycles 1, 3, and 6. The dose of pinometostat will be escalated following a standard 3+3 design, and the primary endpoint for the phase Ib portion will be safety and tolerability. The primary endpoint for the phase II will be response to combination therapy as defined by the 2017 European Leukemia Network guidelines. Accrual will proceed as per a Simon two-stage minimax design. Integrative correlative analyses will include genomics, changes in DOT1L-mediated methylation by H3K79 ELISA, and qPCR of HOXA9 and Meis1. PK studies and azacitidine incorporation and DNA methylation studies will also be performed. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Armstrong:AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; Novartis: Research Funding; Janssen: Research Funding. Rudek:RenovoRX: Research Funding; Taiho: Research Funding; Celgene: Research Funding; Cullinan Apollo: Research Funding. Tallman:Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; ADC Therapeutics: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Cellerant: Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Cellerant: Research Funding; Cellerant: Research Funding; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees.

Description: Cytotoxic lymphocytes, which include CD4+ and CD8+ T cells as well as NK cells, use the granule exocytosis pathway to kill virus-infected and tumor cells. Previous studies have characterized the expression of many genes in this pathway (e.g. perforin, granzyme A, granzyme B, etc.), although no reagent has been developed to measure granzyme C protein at the single-cell level. The murine granzyme C gene, orthologous to human granzyme H, lies 24.2Kb directly downstream from granzyme B in the granzyme B gene cluster. Recombinant granzyme C rapidly induces target cell death in a manner distinct from granzyme A- or B-induced death. To further understand the regulation and function of murine granzyme C, we developed a granzyme C-specific monoclonal antibody and used flow cytometry to examine the expression of granzyme C in resting and activated murine cytotoxic lymphocyte compartments. Naive CD4+ and CD8+ T cells express little or no granzyme C (0.1 + 0.1% of cells are positive). After activation of splenocytes with plate-bound CD3 and CD28 agonistic antibodies for 4 days, a small percentage of CD4+ (2.3 + 1.0% positive) and CD8+ (6.6 + 0.3% positive) T cells express granzyme C. However, only 24 hours later, almost all CD4+ (96.7 + 2.7%) and CD8+ (98.7 + 1.4%) T cells express granzyme C. Granzyme B co-staining revealed that granzyme B is detectable in both CD4+ and CD8+ T cells at least 48 hours before granzyme C is expressed. Furthermore, we employed a fully mismatched GVHD mouse model in order to examine T cell expression of granzymes B and C in vivo; similarly, granzyme B expression preceded granzyme C by at least 48 hours. In addition, CD4+Foxp3+ regulatory T cells also expressed granzyme C in this model. Murine NK cell granzyme C mRNA expression was assessed using Affymetrix microarrays (MOE430v2) at rest and following IL-15 activation. Resting NK cells express minimal granzyme C mRNA (mean gzmC probe set signal intensity + SD [n=3]): 356 + 202, which increased after IL-15 activation as follows: 4,180 + 1,106 (day 1), 60,788 + 8,455 (day 2), 167,448 + 26,398 (day 4), and 178,451+14,037 (day 6). Utilizing our granzyme C-specific mAb, we showed that very few resting murine NK cells express granzyme C protein (2.3 + 0.4% positive). Consistent with the mRNA data, the percentage of granzyme C-expressing NK cells was substantially increased after 3 days of IL-15 activation (88.8 + 5.2% positive). In contrast, granzyme B mRNA levels are high in resting NK cells (41,208 + 2,534), and increase only incrementally with IL-15 treatment (Fehniger et al., Immunity 2007 Jun;26(6):798–811). Granzyme B protein expression is controlled at a post-transcriptional level in NK cells, whereas granzyme C expression is transcriptionally regulated. Taken together, our findings show that the mouse granzyme B and C genes, despite being only 24.2Kb apart, are activated in cytotoxic lymphocytes with different kinetics; moreover, granzyme B and C protein abundance is controlled by completely different mechanisms in NK cells. These data suggest that the granzyme genes are differentially regulated in lymphocyte compartments, and that this regulation may be relevant for how cytotoxic lymphocytes function.

Description: Key Points LSD1 inhibition induces a global increase in chromatin accessibility, whereas DOT1L inhibition induces global decreases in accessibility. Perturbation of PU.1 and C/EBPα expression renders AML cells more resistant to LSD1 inhibition.

Description: Prior studies have shown that the cell of origin of acute myeloid leukemia (AML) initiation is an important determinant of therapeutic sensitivity. MLL-rearranged leukemias in which the fusion is acquired in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and express high levels of the oncogenic transcription factor, Evi1, when compared to leukemias which are initiated in granulocyte-macrophage progenitors (GMP). However, the mechanisms governing how cell-of-origin modulates therapeutic response have not been delineated. Here, we describe a functional link that ties therapeutic sensitivity of MLL-AF9 leukemias to both chemotherapy and pharmacologic inhibitors of lysine-specific demethylase 1 (LSD1) - currently under investigation in clinical trials - to AML cell of origin via a novel mechanism that modulates p53 protein stability and activity through Evi1. This Evi1-dependent mechanism revealed a therapeutic vulnerability in the resistant HSC-derived leukemias that could be targeted with the BCL2 inhibitor venetoclax, which overcame resistance when combined with LSD1 inhibition. Murine HSC-derived MLL-AF9 leukemias exhibited markedly reduced sensitivity to the LSD1 inhibitor, IMG-7289, when compared to GMP-derived MLL-AF9 leukemias, in vitro and in vivo. Consistent with previously published reports, HSC-derived leukemias exhibited several hundred-fold higher expression of EVI1 mRNA relative to GMP-derived leukemias in both mouse and human MLL-rearranged leukemia models; the differential EVI1 expression mirrors expression patterns of this key hematopoietic regulator in normal HSCs and GMPs. H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that the EVI1 locus is silenced by Polycomb repression in GMP-derived leukemias, and this repression was also observed in EVI1low primary AML patient samples. Knockdown of Evi1 via shRNA sensitized HSC-derived leukemias to LSD1 inhibition. In vitro assays revealed induction of apoptosis in GMP-derived leukemias - but not in HSC-derived leukemias - after treatment with IMG-7289. HSC-derived leukemias also exhibited decreased apoptotic priming assessed through functional BH3 profiling assays as well as blunted p53 transcriptional output. These data suggested that cell-of-origin led to differential p53 activity and therapeutic response in AMLs driven by the same fusion gene initiated from different stem/progenitor populations. The diminished p53 activity in HSC-derived leukemias was associated with reduced p53 protein abundance both at steady-state and after LSD1 inhibitor treatment compared to GMP-derived leukemias. Quantification of p53 mRNA did not show differential p53 expression, including after LSD1 inhibition, in HSC vs. GMP-derived leukemias implicating post-transcriptional regulatory mechanisms that underlie this cell-of-origin mediated phenotype. We found that modulation of Evi1 expression resulted in altered p53 protein stability: specifically, (1) shRNA-mediated knockdown of Evi1 in HSC-derived leukemias increased p53 protein stability and (2) overexpression of Evi1 blunted doxorubicin-induced p53 protein stability. Moreover, p53 loss-of-function in Evi1low GMP-derived MLL-AF9 leukemias induced resistance to LSD1 inhibition. By contrast, Evi1high HSC-derived leukemias exposed to the BCL2 inhibitor venetoclax in vivo were sensitized to LSD1 inhibition, resulting in enhanced apoptosis and greater reductions in disease burden, observations that we observed in patients with Evi1high AML treated with venetoclax. Our findings describe how the cell of origin of p53 wild-type cancers can differentially modulate p53 function and therapeutic response and provide a mechanistic rationale for therapies aimed to circumvent this resistance mechanism. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Goldberg:Celgene: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding; American Society of Clinical Oncology: Research Funding; Abbvie: Consultancy; Abbvie: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria. Stein:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Cellerant: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Biosight: Research Funding; Biosight: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Biosight: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lowe:ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership; Blueprint Medicines: Consultancy, Equity Ownership. Rienhoff:Imago Biosciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armstrong:AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; Novartis: Research Funding; Janssen: Research Funding.

Description: Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B–deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of granzyme B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.

Description: NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P

Description: NK-dependent clearance of RMAS lymphoma and B16 melanoma (MHC Ilow) has been demonstrated in NK cell-deficient mice (Kim S et al, PNAS. 2000 Mar; 97(6): 2731–6). We recently investigated the roles of granzyme (Gzm) A and B in NK-dependent clearance of RMAS and B16 cells. The survival curves following intravenous injection of 2x105 RMAS cells are shown in the Figure; similar results were found with B16 cells. All granzyme AxB-deficient mice died within 6 weeks. Survival of granzyme A-deficient mice was similar to that of WT mice. Surprisingly, granzyme B-deficient mice were more resistant than WT mice to these tumor challenges. Previously, our laboratory demonstrated that human regulatory T (Treg) cells can use the granule exocytosis pathway to kill a variety of autologous immune cells in vitro (Grossman WJ et al, Immunity. 2004 Oct; 21(4): 589–601). Based on these results, we hypothesized that these tumor cell lines may induce granzyme B expression in Tregs, which in turn suppress the function of the NK cells responsible for clearing the tumors. Indeed, flow cytometric studies revealed that granzyme B (but not granzyme A) was highly expressed in 10–30% of CD4+/FoxP3+ Tregs found in the tumor environment (i.e. ascites fluid, tumor-infiltrated livers, or lungs). In contrast, very few granzyme B-expressing cells (

Description: Plasmacytoid dendritic cells (pDC) are the principal natural type I interferon producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN) and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia (CMML). The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here we characterize AML patients with pDC expansion (pDC-AML), which we observe in approximately 5% of AML. pDC-AML often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (〉70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, and originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells towards pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.