Murine Acute Myeloid Leukemia Cells Research Articles

The E3 ubiquitin ligase Herc1 modulates the response to nucleoside analogs in acute myeloid leukemia

AbstractFor several decades, induction therapy with nucleoside analogs, in particular cytarabine (Ara-C) and, to a lesser extent, fludarabine, has been the standard of care for patients diagnosed with acute myeloid leukemia (AML). However, the antitumor efficacy of nucleoside analogs is often limited by intrinsic and acquired drug resistance, thereby leading to poor therapeutic response and suboptimal clinical outcomes. In this study, we used genome-wide CRISPR-based pharmacogenomic screening to map the genetic factors that modulate the response to nucleoside analogs in AML and identified the E3 ubiquitin ligase, Herc1, as a key modulator of Ara-C response in mouse AML models driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1, both in vitro and in vivo. Loss of HERC1 enhanced nucleoside analog–induced cell death in both murine and human AML cell lines by compromising cell cycle progression. In-depth proteomic analysis and subsequent validation identified deoxycytidine kinase as a novel target of Herc1 in both mouse AML models. We observed that HERC1 is overexpressed in AML when compared with other cancer types and that higher HERC1 expression was associated with shorter overall survival in patients with AML in the The Cancer Gene Atlas program (TCGA) and BEAT-AML cohorts. Collectively, this study highlights the importance of HERC1 in the response of AML cells to nucleoside analogs, thereby establishing this E3 ubiquitin ligase as a novel predictive biomarker and potential therapeutic target for the treatment of AML.

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway

AbstractAcute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

Hematopoietic Stem and Progenitor Cells Acquire an Inflammatory Memory of the Acute Myeloid Leukemia Niche

Recent studies suggest that acute myeloid leukemia (AML) generates inflammation in the bone marrow (BM) niche to an extent that compromises the function of healthy hematopoietic stem and progenitor cells (HSPC) and impacts patient prognosis. AML xenograft studies have identified several pro-inflammatory mediators (IL-6, IL-1b) that are secreted into the BM niche by leukemic blasts. Prior work by our laboratory also showed contributions involving other AML-secreted factors, in regulating HSPCs in the leukemic niche. HSPC are cellular components of the innate immune system, but the full extent of their inflammatory adaptation by AML is unknown. To overcome some of the experimental constraints inherent in the xenograft approach, we first established non-conditioned congenic grafts of C1498 murine AML cells in C57B/6 recipients. Assessment of the BM revealed that, even at low AML burden, these animals contained a range of elevated pro-inflammatory factors. Single cell RNA-Seq studies of healthy HSPCs, obtained concurrently at sacrifice confirmed an inflammatory transcriptional signature in HSC and MPP populations, with GSEA enrichment for interferon-alpha and -gamma signaling pathways. These observations led us to hypothesize that AML-experienced (inflammation-primed) healthy HSPCs may acquire a durable long-term inflammatory memory that shapes their response to subsequent stimulation during relapse or infection. To test this hypothesis, we adoptively transferred FACS-sorted healthy AML-experienced HSPCs from C1498-engrafted mice (HSPC AML) into secondary recipients. HSPCs transplanted from PBS-injected recipients (HSPC PBS) serve as controls. To assess whether HSPC AML would respond to immune challenge more aggressively compared to HSPC PBS, we exposed these animals with a single-injection of lipopolysaccharide (LPS), followed by tissue harvest and analysis after 24 hours. Bulk RNA-Seq analysis of HSPC AML from the LPS-challenged recipients showed amplified GSEA signatures for tumor necrosis factor- and NF-kB signaling pathways compared to HSPC PBS. In addition, LPS-challenged HSPC AML showed amplified expression by several krüppel-like factors (KLF) genes. Analysis for transposase-accessible chromatin loci (ATAC-Seq), concurrently obtained from LPS-challenged HSPC AML echoed the enrichment of KLF motifs as well. Collectively, the C1498 model suggests that initial AML exposure programs HSPC AML for an amplified inflammatory and differentiation program. To further ascertain disease relevance and reproducibility we turned to a doxycycline-inducible (i)MLL-AF9 AML model. Given the low leukemic burden in the BM of C1498-grafted animals, we wanted to test the hypothesis that secreted factors accounted, at least in part, for the inflammatory memory. Indeed, injection of AML secreted factors from iMLL-AF9 models revealed a strong inflammatory signature in bulk RNA-Seq studies. To determine if innate immune priming in HSPC can be directly mediated by AML crosstalk, we exposed ex vivo-expanded HSPCs to purified secreted factors (SF AML) from (i)MLL-AF9 AML blasts, followed by a secondary inflammatory challenge with LPS seven days later. Gene expression analysis of SF AML-experienced LPS-challenged HSPCs confirmed myeloid-skewed progenitor differentiation and increased expression of inflammatory targets. Together, our data show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory legacy whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our experiments phenocopy observations of trained immunity in other experimental systems, and suggest the acquisition of an innate immune memory in the AML niche. Immune priming may impact the role of these HSPC in the context of inflammation associated with aging, or clones with predisposing molecular lesions such as TET2 or DNMT3A.

Leukemic Cell-Intrinsic Innate Nucleic Acid Receptor Signaling Governs Efficacy of Hypomethylating Agents and Immunosurveillance in AML

Introduction: Hypomethylating agents (HMA), such as Azaciditine (AZA), are a long-established treatment option for acute myeloid leukemia (AML). Besides epigenetic reprogramming of malignant cell clones, HMAs are recognized to facilitate antineoplastic immunity. In vitro assessments in solid tumor models suggest that a major mechanism underlying AZA-triggered immune responses may be the activation of innate nucleic acid sensing pathways. As such, the RNA- and DNA-sensing receptor systems, RIG-I/MDA-5 and cGAS/STING - and subsequent type I interferon (IFN-I) signaling seem to be triggered by the transcriptional upregulation of otherwise DNA-hypermethylated and thus silenced endogenous retroviruses (ERVs). In AML, it is unknown whether similar processes are involved and/or by which molecular mechanisms, HMAs modulate anti-leukemic immunity against AML blasts, and how these processes are affected during acquired HMA resistance. Methods: Murine (c1498 and Wehi-3B) and human (MOLM-13) AML cell lines were exposed to AZA in vitro and analyzed for expression of ERVs. Nucleic acid receptor sensing pathways and IFN-I genes are not usually DNA methylated by AZA at promoter regions. However, the re-expression of endogenous nucleic acids or ERVs could trigger their activation. Protein and transcriptional expression of major components of nucleic acid receptor sensing pathways and IFN-I genes were, therefore, analysed. To understand the importance of AML cell-intrinsic nucleic acid receptor signaling in HMA-induced immunosurveillance, syngeneic c1498 and Wehi-3B AML models were employed. Mice received 2 cycles of 2.5mg/kg AZA (6 days on, 5 days off, 6 days on) intraperitoneally to mimic the principal treatment course used in patients. To address the contribution of the nucleic acid-sensing pathways to AZA treatment efficacy in vitroand in vivo, AML cells with acquired AZA resistance (AZA-R) or gene engineered AML cells deficient in RNA receptors (RIG-I/MDA-5), or the RNA- and DNA-sensing adapter proteins (MAVS and STING) were employed. Results: A targeted ERV screen revealed upregulation of various ERV transcripts in c1498 and Wehi-3B cells and was similarly observed in MOLM-13 AML cells following AZA exposure in vitro. Increased RNA transcript and protein expression of RIG-I/MAVS and (to a lesser degree) STING was observed, resulting in activation of downstream constituents including higher phosphorylation of the transcription factors IRF3/7 and NF-kB, IFN-I, and other ISGs. This was abrogated in RIG-I and MAVS, but not STING, deficient murine and human AML cells. Interestingly, expression of RIG-I/MDA-5, STING and all other downstream constituents, but not MAVS, were diminished in AZA-R AML cells. In mice harboring MAVS deficient AML (c1498), HMA treatment failed to show anti-leukemic activity resulting in rapid disease progress and death of recipient animals. Loss of STING in leukemic cells also impacted AZA treatment efficacy, although to a much less pronounced degree. These findings were similarly reproduced with Wehi-3B AML-engrafted mice. Following antibody-mediated depletion of T cells but not NK cells, mice rapidly succumbed to AML disease and demonstrated no anti-leukemic effect of AZA treatment. CyTOF and flow cytometric analysis also revealed increased bone marrow and spleen infiltration as well as activation of CD4 +/CD8 + T cells, NK cells, and MHC-II + APCs/Myeloid cells in AZA treated mice bearing wildtype AML, compared to untreated. This was not observed in AZA-treated mice bearing MAVS-deficient or AZA-R AML. Conclusion: Our findings indicate that nucleic acid receptor signaling (specifically RIG-I/MAVS) within leukemic cells is imperative for AZA efficacy and immunosurveillance in AML. Whether these RNA-sensing receptors are triggered by ERVs or alternate (endogenous) RNAs remains to be determined. Elucidating the molecular mechanisms of how nucleic acid sensors within leukemic blasts shape treatment efficacy of AZA may guide development of systematic immunotherapeutic strategies for AZA-R AML by targeted combinatorial approaches.

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

AMPK activation induces immunogenic cell death in AML

AbstractSurvival of patients with acute myeloid leukemia (AML) can be improved by allogeneic hematopoietic stem cell transplantation (allo-HSCT) because of the antileukemic activity of T and natural killer cells from the donor. However, the use of allo-HSCT is limited by donor availability, recipient age, and potential severe side effects. Similarly, the efficacy of immunotherapies directing autologous T cells against tumor cells, including T-cell recruiting antibodies, chimeric antigen receptor T-cell therapy, and immune checkpoint inhibitors are limited in AML because of multiple mechanisms of leukemia immune escape. This has prompted a search for novel immunostimulatory approaches. Here, we show that activation of adenosine 5′-monophosphate–activated protein kinase (AMPK), a master regulator of cellular energy balance, by the small molecule GSK621 induces calreticulin (CALR) membrane exposure in murine and human AML cells. When CALR is exposed on the cell surface, it serves as a damage-associated molecular pattern that stimulates immune responses. We found that GSK621-treated murine leukemia cells promote the activation and maturation of bone marrow–derived dendritic cells. Moreover, vaccination with GSK621-treated leukemia cells had a protective effect in syngeneic immunocompetent recipients bearing transplanted AMLs. This effect was lost in recipients depleted of CD4/CD8 T cells. Together, these results demonstrate that AMPK activation by GSK621 elicits traits of immunogenic cell death and promotes a robust immune response against leukemia. Pharmacologic AMPK activation thus represents a new potential target for improving the activity of immunotherapy in AML.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [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 525.

Dipeptidylpeptidase 4 promotes survival and stemness of acute myeloid leukemia stem cells.

Leukemic-stem-cell-specific targeting may improve the survival of patients with acute myeloid leukemia (AML) by avoiding the ablative effects of standard regimens on normal hematopoiesis. Herein, we perform an unbiased screening of compounds targeting cell surface proteins and identify clinically used DPP4 inhibitors as strong suppressors of AML development in both murine AML models and primary human AML cells xenograft model. We find in retrovirus-induced AML mouse models that DPP4-deficient AML cell-transplanted mice exhibit delay and reversal of AML development, whereas deletion of DPP4 has no significant effect on normal hematopoiesis. DPP4 activates and sustains survival of AML stem cells that are critical for AML development in both human and animal models via binding with Src kinase and activation of nuclear factor κB (NF-κB) signaling. Thus, inhibition of DPP4 is a potential therapeutic strategy against AML development through suppression of survival and stemness of AML cells.

The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

Modeling Flotetuzumab-Associated CRS in AML Using inVitro and inVivo Preclinical Models

Introduction: Flotetuzumab (FLZ) is a bispecific CD123 x CD3 DART® molecule that is being investigated for the treatment of relapsed and refractory acute myeloid leukemia (AML). FLZ induces artificial immunological synapses between CD3 on cytotoxic T-cells and CD123, which is expressed on AML blasts along with off-target hematopoietic and non-hematopoietic tissues. T-cell activation and expansion requires cytokine release from both innate and adaptive immune cells, which when excessive, can result in cytokine release syndrome (CRS), a potentially life-threatening toxicity that manifests with fever, hypotension, organ dysfunction, respiratory failure, and/or coagulopathy. FLZ-mediated anti-leukemia efficacy is often limited by CRS. Therefore, developing pre-clinical models by which to better understand the mechanisms of CRS induction, including collaborating cell types that produce IL-6 and other inflammatory cytokines, is essential to understanding the biology of CRS and developing mechanistically plausible preventative and treatment strategies. MacroGenics has developed a murine genetic knock-in model that expresses a chimeric murine CD3 epsilon (CD3e) chain bearing the human CD3e epitope recognized by FLZ (hCD3e). These mice are immune competent, maintain normal signaling through the T-cell receptor and expand and secrete cytokines upon binding to FLZ and human CD123. To validate this model, we explore whether T-cells harvested from the spleens of immune competent C57Bl/6 hCD3e mice will bind FLZ and kill human CD123-expressing target cells in vitro. Methods: A murine AML cell line (AML1-GFP FLT3-ITD, DNMT3AR878H) was transduced with human CD123 (hCD123) using a lentiviral vector construct and single cell clones expressing high levels of hCD123 were expanded (AMLGFP-hCD123). Spleens from hCD3eHOM C57Bl/6 mice were harvested for isolation of T-lymphocytes via negative immunomagnetic selection on an AutoMACS machine. In vitro killing assays were set up using purified C57Bl/6 hCD3eHOM T-cells and two clones of AMLGFP-hCD123 cells. The effector to target ratio was 1:1, with 2.5x105 cells per well in a 24-well plate. Flow cytometry/FACS was then performed at 24 hours to assess killing of AMLGFP-hCD123 clones 4 and 14 and T-cell subset number and phenotype. Antibodies to the following antigens were used for FACS: mCD4, hCD3 (epitope SP38), mCD25, mCD8, mCD44, mCD62L, hCD123, mCD3, mCD127, mCD69, and mCD45, in addition to endogenous GFP. Results: At 24 hours, target cell killing of clones 4 and 14 was noted (Figure 1). Compared to the controls treated with FLZ or T-cells alone, targets treated with T-cells + FLZ showed a 2.3- and 2.5-fold decrease in the number of AMLGFP-hCD123 targets in clones 4 and 14, respectively, at 24 hours (Figure 1). In terms of T-cell phenotypic changes, the percentage and MFI expression of activation markers, namely CD25 and CD69, increased for CD4+ and CD8+ subsets in the treatment group compared to controls (data not shown). T-cell subsets were characterized as naïve (CD62L+/CD44-), central memory (CD62L+/CD44+), and effector memory (CD62L-/CD44+), as shown in Figure 2. For CD4+ and CD8+ subsets co-cultured with targets, the addition of FLZ induced phenotypic changes away from naïve and towards central and effector memory subsets (Figure 2). In the two control groups, naïve T-cells predominated at the 24-hour time point (Figure 2). Conclusions: The in vitro killing assay shows that T-cells from the spleens of immune competent C57Bl/6 hCD123eHOM mice recognize FLZ and hCD123 on transduced murine leukemia cells. Co-culture with T-cells + target cells + FLZ showed killing at 24 hours compared to controls. T-cell activation and proliferation were noted based on cell surface markers. The shift in phenotype from naïve to central and effector memory subsets suggests T-cell expansion upon leukemia antigen recognition and portends killing of target cells. Pending in vivo studies will evaluate if FLZ can activate endogenous T cells, induce CRS, and kill AMLGFP-hCD123 targets in the huCD3eHOM mice. If successful, we will use the huCD3eHOM model to test the efficacy of various therapies to mitigate CRS and preserve FLZ-mediated T-cell cytotoxicity against AMLGFP-hCD123 targets in huCD3eHOM mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation

AbstractPatients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell–mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Bcr-TMP, a Novel Nanomolar-Active Compound That Exhibits Both MYB- and Microtubule-Inhibitory Activity.

Simple SummaryRecent work has identified the transcription regulator MYB as an interesting therapeutic target for the treatment of certain leukemias and other cancers that are dependent on deregulated MYB activity, such as acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here we report the identification and characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a novel highly active MYB inhibitory compound. We show that nanomolar concentrations of Bcr-TMP are sufficient to down-regulate the expression of MYB target genes and induce both cell-death and differentiation in AML cell lines. Importantly, Bcr-TMP also and exerts stronger anti-proliferative effects on MYB-addicted primary AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Preliminary work shows that Bcr-TMP acts through p300, a protein interacting with MYB and stimulating its activity. Interestingly, Bcr-TMP has an additional activity as an anti-microtubule agent. Overall, Bcr-TMP is an interesting compound that warrants further research to understand its mechanism of action and its therapeutic potential for MYB-dependent malignancies.Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here, we present the initial characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a nanomolar-active MYB-inhibitory compound identified in a screen for novel MYB inhibitors. Bcr-TMP affects MYB function in a dual manner by inducing its degradation and suppressing its transactivation potential by disrupting its cooperation with co-activator p300. Bcr-TMP also interferes with the p300-dependent stimulation of C/EBPβ, a transcription factor co-operating with MYB in myeloid cells, indicating that Bcr-TMP is a p300-inhibitor. Bcr-TMP reduces the viability of AML cell lines at nanomolar concentrations and induces cell-death and expression of myeloid differentiation markers. It also down-regulates the expression of MYB target genes and exerts stronger anti-proliferative effects on MYB-addicted primary murine AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Surprisingly, we observed that Bcr-TMP also has microtubule-disrupting activity, pointing to a possible link between MYB-activity and microtubule stability. Overall, Bcr-TMP is a highly potent multifunctional MYB-inhibitory agent that warrants further investigation of its therapeutic potential and mechanism(s) of action.

Acute myeloid leukemia cell membrane-coated nanoparticles for cancer vaccination immunotherapy

Cancer vaccines are promising treatments to prevent relapse after chemotherapy in acute myeloid leukemia (AML) patients, particularly for those who cannot tolerate intensive consolidation therapies. Here, we report the development of an AML cell membrane-coated nanoparticle (AMCNP) vaccine platform, in which immune-stimulatory adjuvant-loaded nanoparticles are coated with leukemic cell membrane material. This AMCNP vaccination strategy stimulates leukemia-specific immune responses by co-delivering membrane-associated antigens along with adjuvants to antigen-presenting cells. To demonstrate that this AMCNP vaccine enhances leukemia-specific antigen presentation and T cell responses, we modified a murine AML cell line to express membrane-bound chicken ovalbumin as a model antigen. AMCNPs were efficiently acquired by antigen-presenting cells in vitro and in vivo and stimulated antigen cross-presentation. Vaccination with AMCNPs significantly enhanced antigen-specific T cell expansion and effector function compared with control vaccines. Prophylactic vaccination with AMCNPs enhanced cellular immunity and protected against AML challenge. Moreover, in an AML post-remission vaccination model, AMCNP vaccination significantly enhanced survival in comparison to vaccination with whole leukemia cell lysates. Collectively, AMCNPs retained AML-specific antigens, elicited enhanced antigen-specific immune responses, and provided therapeutic benefit against AML challenge.

Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy.We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival.Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment.Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. DisclosuresNeel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Corresponding author: Dr. Simon. C. Robson (srobson@bidmc.harvard.edu).Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model.Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions.Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I).Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models.Disclosures: No relevant conflicts of interest to declare.

Runt-Related Transcription Factor 3 Promotes Acute Myeloid Leukemia Progression.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with high relapse/refractory rate. Genetic and epigenetic abnormalities are driving factors for leukemogenesis. RUNX1 and RUNX2 from the Runt-related transcription factor (RUNX) family played important roles in AML pathogenesis. However, the relationship between RUNX3 and AML remains unclear. Here, we found that RUNX3 was a super-enhancer-associated gene and highly expressed in AML cells. The Cancer Genome Atlas (TCGA) database showed high expression of RUNX3 correlated with poor prognosis of AML patients. We observed that Runx3 knockdown significantly inhibited leukemia progression by inducing DNA damage to enhance apoptosis in murine AML cells. By chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we discovered that RUNX3 in AML cells mainly bound more genes involved in DNA-damage repair and antiapoptosis pathways compared to that in normal bone marrow cells. Runx3 knockdown obviously inhibited the expression of these genes in AML cells. Overall, we identified RUNX3 as an oncogene overexpressed in AML cells, and Runx3 knockdown suppressed AML progression by inducing DNA damage and apoptosis.

Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells.

Simple SummaryDespite all of the advancements made in recent years in the treatment of acute myeloid leukemia (AML), long-term survival is achieved by only 30–40% of AML patients. Thus, new therapeutic strategies are strongly needed. This work confirms the increase in oxidative phosphorylation upon cytarabine resistance in AML murine cells, reinforcing the interest of targeting it. In addition, it identifies a new role of the first complex of the electron transport chain (ETCI) in the regulation of the immune checkpoints PD-L1 and CD39 in murine and human leukemic cells. Thus, this work opens the door to the evaluation of ETCI inhibitors in combination with immunotherapy.Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the anti-tumor response. The modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve the therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as ectonucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemo-resistance and a poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased the OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that while AraC induced an immune response regulation by increasing CD39 expression and by reinforcing the interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.