Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Ferritin heavy/light chain (FTH1/FTL) expression, serum ferritin levels, and their functional as well as prognostic roles in acute myeloid leukemia.

OBJECTIVES: We previously reported the prognostic value of serum ferritin in younger patients with intermediate-risk acute myeloid leukemia (AML). The aims of this study were to confirm this finding in a larger cohort regardless of age and prognostic subgroups, to explore the expression and functional role of ferritin in AML cells as well as the regulation of serum ferritin levels in AML patients. PATIENTS/MATERIALS/METHODS: Serum ferritin levels at diagnosis were collected in a cohort of 525 patients treated by intensive chemotherapy. In silico, in vitro, and in vivo analyses were conducted to assess the pattern of expression and functional role of FTH1 and FTL in AML. RESULTS: We confirmed the independent prognostic value of serum ferritin. In transcriptomic databases, FTH1 and FTL were overexpressed in AML and leukemic stem cells compared to normal hematopoietic stem cells. The gene signature designed from AML patients overexpressing FTH1 revealed a significant enrichment in genes of the immune and inflammatory response including Nf-KB pathway, oxidative stress, or iron pathways. This gene signature was enriched in cytarabine-resistant AML cells in a patient-derived xenograft model. FTH1 protein was also overexpressed in patient's samples and correlated with the in vitro cytotoxic activity of cytarabine. Lastly, we demonstrated that chemotherapy induced an inflammatory response including a significant increase in serum ferritin levels between day 1 and 8 of induction chemotherapy that was blocked by dexamethasone. CONCLUSION: Ferritin is deregulated in most AML patients likely through inflammation, associated with chemoresistance, and could represent a new therapeutic target.

Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells.

Internal tandem duplication of the Fms-like tyrosine kinase-3 receptor (FLT3) internal tandem duplication (ITD) is found in 30% of acute myeloid leukemia (AML) and is associated with a poor outcome. In addition to tyrosine kinase inhibitors, therapeutic strategies that modulate the expression of FLT3-ITD are also promising. We show that AML samples bearing FLT3-ITD mutations are more sensitive to proteasome inhibitors than wild-type samples and this sensitivity is strongly correlated with a higher FLT3-ITD allelic burden. Using pharmacologic inhibitors of autophagy, specific downregulation of key autophagy proteins including Vps34, autophagy gene (Atg)5, Atg12, Atg13, biochemical, and microscopy studies, we demonstrated that proteasome inhibitors induced cytotoxic autophagy in AML cells. FLT3-ITD molecules were detectable within autophagosomes after bortezomib treatment indicating that autophagy induction was responsible for the early degradation of FLT3-ITD, which preceded the inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), PI3K/AKT, and STAT5 pathways, and subsequent activation of cell death. Moreover, proteasome inhibitors overcome resistance to quizartinib induced by mutations in the kinase domain of FLT3, suggesting that these compounds may prevent the emergence of mutant clones arising from tyrosine kinase inhibitor treatments. In xenograft mice models, bortezomib stimulated the conversion of LC3-I to LC3-II, indicating induction of autophagy in vivo, downregulated FLT3-ITD protein expression and improved overall survival. Therefore, selecting patients according to FLT3-ITD mutations could be a new way to detect a significant clinical activity of proteasome inhibitors in AML patients.

Dexamethasone in hyperleukocytic acute myeloid leukemia.

Patients with acute myeloid leukemia and a high white blood cell count are at increased risk of early death and relapse. Because mediators of inflammation contribute to leukostasis and chemoresistance, dexamethasone added to chemotherapy could improve outcomes. This retrospective study evaluated the impact of adding or not adding dexamethasone to chemotherapy in a cohort of 160 patients with at least 50×109 white blood cells. In silico studies, primary samples, leukemic cell lines, and xenograft mouse models were used to explore the antileukemic activity of dexamethasone. There was no difference with respect to induction death rate, response, and infections between the 60 patients in the dexamethasone group and the 100 patients in the no dexamethasone group. Multivariate analysis showed that dexamethasone was significantly associated with improved relapse incidence (adjusted sub-HR: 0.30; 95% CI: 0.14-0.62; P=0.001), disease-free survival (adjusted HR: 0.50; 95% CI: 0.29-0.84; P=0.010), event-free survival (adjusted HR: 0.35; 95% CI: 0.21-0.58; P<0.001), and overall survival (adjusted HR: 0.41; 95% CI: 0.22-0.79; P=0.007). In a co-culture system, dexamethasone reduced the frequency of leukemic long-term culture initiating cells by 38% and enhanced the cytotoxicity of doxorubicin and cytarabine. In a patient-derived xenograft model treated with cytarabine, chemoresistant cells were enriched in genes of the inflammatory response modulated by dexamethasone. Dexamethasone also demonstrated antileukemic activity in NPM1-mutated samples. Dexamethasone may improve the outcome of acute myeloid leukemia patients receiving intensive chemotherapy. This effect could be due to the modulation of inflammatory chemoresistance pathways and to a specific activity in acute myeloid leukemia with NPM1 mutation.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

Mitochondrial fusion is a therapeutic vulnerability of acute myeloid leukemia.

Mitochondrial metabolism recently emerged as a critical dependency in acute myeloid leukemia (AML). The shape of mitochondria is tightly regulated by dynamin GTPase proteins, which drive opposing fusion and fission forces to consistently adapt bioenergetics to the cellular context. Here, we showed that targeting mitochondrial fusion was a new vulnerability of AML cells, when assayed in patient-derived xenograft (PDX) models. Genetic depletion of mitofusin 2 (MFN2) or optic atrophy 1 (OPA1) or pharmacological inhibition of OPA1 (MYLS22) blocked mitochondrial fusion and had significant anti-leukemic activity, while having limited impact on normal hematopoietic cells ex vivo and in vivo. Mechanistically, inhibition of mitochondrial fusion disrupted mitochondrial respiration and reactive oxygen species production, leading to cell cycle arrest at the G0/G1 transition. These results nominate the inhibition of mitochondrial fusion as a promising therapeutic approach for AML.

CD36 Drives Metastasis and Relapse in Acute Myeloid Leukemia.

Identifying mechanisms underlying relapse is a major clinical issue for effective cancer treatment. The emerging understanding of the importance of metastasis in hematologic malignancies suggests that it could also play a role in drug resistance and relapse in acute myeloid leukemia (AML). In a cohort of 1,273 AML patients, we uncovered that the multifunctional scavenger receptor CD36 was positively associated with extramedullary dissemination of leukemic blasts, increased risk of relapse after intensive chemotherapy, and reduced event-free and overall survival. CD36 was dispensable for lipid uptake but fostered blast migration through its binding with thrombospondin-1. CD36-expressing blasts, which were largely enriched after chemotherapy, exhibited a senescent-like phenotype while maintaining their migratory ability. In xenograft mouse models, CD36 inhibition reduced metastasis of blasts and prolonged survival of chemotherapy-treated mice. These results pave the way for the development of CD36 as an independent marker of poor prognosis in AML patients and a promising actionable target to improve the outcome of patients. SIGNIFICANCE: CD36 promotes blast migration and extramedullary disease in acute myeloid leukemia and represents a critical target that can be exploited for clinical prognosis and patient treatment.

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy.

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD).

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia.

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3). Previous studies have demonstrated that AML cells expressing FLT3-internal tandem duplication (ITD) are more sensitive to the proteasome inhibitor bortezomib (Bz) than FLT3 wild-type cells, with this cytotoxicity being mediated by autophagy (Atg). Here, we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM-14 leukemic cells expressing the FLT3-ITD mutation, but not in OCI-AML3 leukemic cells with wild-type FLT3. Chemical inhibition of Atg with bafilomycin A simultaneously blocked proteaphagy and resulted in the accumulation of the p62 Atg receptor in Bz-treated MOLM-14 cells. The use of ubiquitin traps revealed that ubiquitin plays an important role in proteasome-Atg cross-talk. The p62 inhibitor verteporfin blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3-ITD in Bz-treated MOLM-14 cells. Both Atg inhibitors enhanced Bz-induced apoptosis in FLT3-ITD-driven leukemic cells, highlighting the therapeutic potential of these treatments.

AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia.

The treatment of acute myeloid leukemia (AML) remains a challenge especially among the elderly. The Bcl-2 inhibitor venetoclax recently showed significant survival benefits in AML patients when combined to low-dose cytarabine or azacitidine. Bcl-2 inhibition initiate mitochondrial apoptosis, but also respiration and cellular ATP production in AML. AMP-Activated Protein Kinase (AMPK) is a central energy sensor activated by increased AMP:ATP ratio to restore the cellular energy balance. Unexpectedly, we observed that venetoclax inhibited AMPK activity through caspase-dependent degradation of AMPK subunits in AML cells. On the other hand, genetic models of AMPK invalidation and re-expression suggested that AMPK participated to the early stages of apoptotic response through a negative regulation of multi-domain anti-apoptotic effectors such as Mcl-1 or Bcl-xL. Together our results suggested a new link between AMPK and Bcl-2-dependent mitochondrial apoptosis that participated to the anti-leukemic activity of venetoclax in AML.

Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells.

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

Mitochondrial metabolism supports resistance to IDH mutant inhibitors in acute myeloid leukemia.

Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid ß-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors.

Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies.

Dendrogenin A synergizes with Cytarabine to Kill Acute Myeloid Leukemia Cells In Vitro and In Vivo.

Dendrogenin A (DDA) is a mammalian cholesterol metabolite that displays potent antitumor properties on acute myeloid leukemia (AML). DDA triggers lethal autophagy in cancer cells through a biased activation of the oxysterol receptor LXRß, and the inhibition of a sterol isomerase. We hypothesize that DDA could potentiate the activity of an anticancer drug acting through a different molecular mechanism, and conducted in vitro and in vivo combination tests on AML cell lines and patient primary tumors. We report here results from tests combining DDA with antimetabolite cytarabine (Ara-C), one of the main drugs used for AML treatment worldwide. We demonstrated that DDA potentiated and sensitized AML cells, including primary patient samples, to Ara-C in vitro and in vivo. Mechanistic studies revealed that this sensitization was LXRß-dependent and was due to the activation of lethal autophagy. This study demonstrates a positive in vitro and in vivo interaction between DDA and Ara-C, and supports the clinical evaluation of DDA in combination with Ara-C for the treatment of AML.

Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites.

Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid ß-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.

Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia.

Relapses driven by chemoresistant leukemic cell populations are the main cause of mortality for patients with acute myeloid leukemia (AML). Here, we show that the ectonucleotidase CD39 (ENTPD1) is upregulated in cytarabine-resistant leukemic cells from both AML cell lines and patient samples in vivo and in vitro. CD39 cell-surface expression and activity is increased in patients with AML upon chemotherapy compared with diagnosis, and enrichment in CD39-expressing blasts is a marker of adverse prognosis in the clinics. High CD39 activity promotes cytarabine resistance by enhancing mitochondrial activity and biogenesis through activation of a cAMP-mediated adaptive mitochondrial stress response. Finally, genetic and pharmacologic inhibition of CD39 ecto-ATPase activity blocks the mitochondrial reprogramming triggered by cytarabine treatment and markedly enhances its cytotoxicity in AML cells in vitro and in vivo. Together, these results reveal CD39 as a new residual disease marker and a promising therapeutic target to improve chemotherapy response in AML. SIGNIFICANCE: Extracellular ATP and CD39-P2RY13-cAMP-OxPHOS axis are key regulators of cytarabine resistance, offering a new promising therapeutic strategy in AML.This article is highlighted in the In This Issue feature, p. 1426.

Oncogenic KIT mutations induce STAT3-dependent autophagy to support cell proliferation in acute myeloid leukemia.

Autophagy is associated with both survival and cell death in myeloid malignancies. Therefore, deciphering its role in different genetically defined subtypes of acute myeloid leukemia (AML) is critical. Activating mutations of the KIT receptor tyrosine kinase are frequently detected in core-binding factor AML and are associated with a greater risk of relapse. Herein, we report that basal autophagy was significantly increased by the KITD816V mutation in AML cells and contributed to support their cell proliferation and survival. Invalidation of the key autophagy protein Atg12 strongly reduced tumor burden and improved survival of immunocompromised NSG mice engrafted with KITD816V TF-1 cells. Downstream of KITD816V, STAT3, but not AKT or ERK pathways, was identified as a major regulator of autophagy. Accordingly, STAT3 pharmacological inhibition or downregulation inhibited autophagy and reduced tumor growth both in vitro and in vivo. Taken together, our results support the notion that targeting autophagy or STAT3 opens up an exploratory pathway for finding new therapeutic opportunities for patients with CBF-AML or others malignancies with KITD816V mutations.