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Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic stem and progenitor cells characterized by profound metabolic dysregulation. Pyrimidine biosynthesis has emerged as a critical metabolic dependency in AML, but clinical translation has been hampered by unacceptable toxicity of current pyrimidine synthesis inhibitors. Since aspartate is an essential nutrient for pyrimidine biosynthesis, we investigated the role of aspartate import via the excitatory amino acid transporter 1 (EAAT1) in AML. We found that EAAT1 is broadly expressed across AML cell lines and patient samples, with enrichment in M4 and M5 subtypes and increasing levels following chemotherapy treatment. Pharmacological inhibition of EAAT1 impaired AML cell viability in vitro , but metabolomic profiling and nutrient rescue experiments showed that these effects were independent of intracellular aspartate levels. Moreover, AML cells cultured in aspartate-free medium maintained proliferation and did not become more sensitive to chemotherapy. EAAT1 inhibition in mice increased bone marrow plasma aspartate levels, confirming inhibition of cellular aspartate uptake, but did not affect growth or chemosensitivity of MLL-AF9-expressing AML cells in vivo . These findings suggest that AML cells possess several complementary mechanisms to support their aspartate requirements and that EAAT1 inhibition does not impair AML growth or response to chemotherapy.

The survival of leukemic cells is significantly influenced by the bone marrow microenvironment, where stromal cells play a crucial role. While there has been substantial progress in understanding the mechanisms and pathways involved in this crosstalk, limited data exist regarding the impact of leukemic cells on bone marrow stromal cells and their potential role in drug resistance. In this study, we identify that leukemic cells prime bone marrow stromal cells towards osteoblast lineage and promote drug resistance. This biased differentiation of stroma is accompanied by dysregulation of the canonical Wnt signaling pathway. Inhibition of Wnt signaling in stroma reversed the drug resistance in leukemic cells, which was further validated in leukemic mice models. This study evaluates the critical role of leukemic cells in establishing a drug-resistant niche by influencing the bone marrow stromal cells. Additionally, it highlights the potential of targeting Wnt signaling in the stroma by repurposing an anthelmintic drug to overcome the microenvironment-mediated drug resistance.

Background Arsenic trioxide (ATO) therapy is highly successful in majority of acute promyelocytic leukemia (APL) patients with PML-RARA fusion oncoprotein. However, after initial response to therapy, 8–10% experienced relapse and became resistant to ATO. ATO-resistant APL cells show elevated BCL2 expression and increased reliance on oxidative phosphorylation (OXPHOS) for survival. Venetoclax (Ven), a BCL2 inhibitor, induces apoptosis by suppressing OXPHOS and increasing mitochondrial reactive oxygen species (ROS). Method Cytotoxic effects of venetoclax were assessed in APL cell lines and primary blasts using CTG assays. Mitochondrial function, biogenesis, and apoptosis were evaluated through Seahorse extracellular flux analysis, flow cytometry–based assays, transmission electron microscopy, and immunoblotting. For mechanistic studies, we carried out genetic knockdown of BCL2 and Beclin-1 and performed co-immunoprecipitation. Proteomic profiling of ATO-resistant cells treated with venetoclax or ATO was conducted using high-resolution LC-MS. Leukemic burden, apoptosis, and mitochondrial ROS were studied in NOD/SCID orthotopic xenografts. An FVB/N-PML-RARA syngeneic model was used to establish in vivo ATO resistance and evaluate OXPHOS, apoptosis, mitochondrial ROS and tumor burden. Results Antiproliferative activity of venetoclax in primary APL blasts ( n  = 25) was in nanomolar concentration (mean = 39 nM; range = 0.015-280 nM). Venetoclax in ATO-resistant cells decreases mitochondrial respiration, increases mitochondrial ROS, and disrupts mitochondrial membrane potential. These events led to cytochrome C release to activate mitochondrial apoptosis. Proteomic profiling using high-resolution Liquid Chromatography-Mass Spectrometry identified differentially expressed proteins, revealing dysregulated pathways associated with apoptosis and autophagy. Venetoclax downregulated anti-apoptotic proteins and degraded PML-RARA. Mechanistically, venetoclax disrupted BCL2/Beclin-1 interaction, releasing Beclin-1 to initiate autophagy by modulating the levels of p62 and LC3-I to LC3-II conversion. Furthermore, in orthotopic xenografts, venetoclax treatment results in the reduction of hCD45+ cells in bone marrow to 4.8% (range: 2.5–5.5%) compared to 9.4% (range: 8.6–10.5%) in vehicle control ( p  < 0.0001). Ven-ATO further reduced this to 3.8% (range: 1.6–4.8%; p  < 0.0001), whereas ATO decreased hCD45+ cells to 8.0% (range: 5.5–12.3%; p  = 0.16). Additionally, the therapeutic efficacy of venetoclax was confirmed using in vivo ATO-resistant FVB/N-PML-RARA model. Conclusions These findings suggest that venetoclax induced apoptosis and autophagy in ATO-resistant cells by increasing mitochondrial stress and disrupting BCL2/Beclin-1 interaction. Graphical abstract

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

Refractory acute myeloid leukemia (AML), defined as failure of two cycles of induction therapy at diagnosis or of one cycle at relapse, represents a subgroup with poor outcomes. Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (fludarabine + cytosine + granulocyte colony-stimulating factor ± idarubicin or mitoxantrone + etoposide) followed by 1-week rest and then reduced-intensity transplant with fludarabine + melphalan. We used the same backbone for this trial (CTRI/2019/02/017505) with the addition of CD56-positive cells from a family donor infused 1 day after the completion of chemotherapy. CD56-positive selection was done using a CliniMACS Prodigy system (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by overnight incubation in autologous plasma with 2 micromolar arsenic trioxide and 500 U/mL of interleukin-2. From February 2019, 14 patients with a median age of 29 years (interquartile range [IQR]: 16.5–38.5) were enrolled in this trial. Six were females. Six had primary refractory AML while eight had relapsed refractory AML. The median CD56-cell dose infused was 46.16 × 106/kg (IQR: 25.06–70.36). One patient withdrew consent after NK cell infusion. Of the 13 patients who proceeded to transplant, five died of immediate post-transplant complications while two did not engraft but were in morphologic leukemia-free state (both subsequently died of infective complications after the second transplant). Of the remaining six patients who engrafted and survived beyond 1 month of the transplant, two developed disease relapse and died. The remaining four patients are alive and relapse free at the last follow-up (mean follow-up duration of surviving patients is 24 months). The 2-year estimated overall survival for the cohort was 28.6% ± 12.1% while the treatment-related mortality (TRM) with this approach was 38.5% ± 13.5%. Haploidentical NK cell therapy as an adjunct to transplant is safe and needs further exploration in patients with AML. For refractory AML, post-transplant NK infusion and strategies to reduce TRM while using pre-transplant NK infusion merit exploration.

Acute myeloid leukemia (AML) remains difficult to cure despite recent advances. Off-target side effects of drugs currently used lead to significant morbidity and mortality. There is recognition that in AML, there is an increased dependence on OXPHOS metabolism, especially in the leukemia stem cell compartment (AML-LSC). It is also recognized that there is potential to exploit this vulnerability to treat AML. Drug re-purposing screens have suggested the potential use of artesunate (ART) to inhibit mitochondrial respiration. We have explored the potential role of ART as an additive agent in treating AML in combination with conventional therapy. Through in-vitro and in-vivo mouse model studies, we demonstrate the mechanism and efficacy of these combinations and their potential to overcome venetoclax resistance. We further demonstrate the specificity of these combinations with minimal off-target effects on normal hematopoietic stem cells (HSC). These observations warrant exploration of the additive role of ART in clinical trials.

The standard‐of‐care for patients with acute promyelocytic leukemia (APL) relapsing after upfront arsenic trioxide (ATO) therapy is not defined. The present study was undertaken to evaluate the safety of addition of bortezomib to ATO in the treatment of relapsed APL based on our previously reported preclinical data demonstrating synergy between these agents. This was an open label, nonrandomized, phase II, single‐center study. We enrolled 22 consecutive patients with relapsed APL. The median age was 26.5 years (interquartile range 17.5 to 41.5). The median time from initial diagnosis to relapse was 23.1 months (interquartile range 15.6 to 43.8). All patients achieved hematological remission at a median time of 45 days (range 40‐63). Nineteen patients were in molecular remission at the end of induction. Grade 3 adverse events occurred in eight instances with one patient requiring discontinuation of therapy for grade 3 neuropathy. Twelve (54.5%) patients underwent autologous transplantation (auto‐SCT) in molecular remission while the rest opted for maintenance therapy. The median follow‐up was 48 months (range 28‐56.3). Of the patients undergoing auto‐SCT, all except one was alive and relapse free at last follow‐up. Of the patients who opted for maintenance therapy, three developed a second relapse. For treatment of APL relapsing after upfront ATO therapy, addition of bortezomib to a standard ATO‐based salvage regimen is safe and effective. This trial was registered at www.clinicaltrials.gov as NCT01950611. The standard‐of‐care for patients with acute promyelocytic leukemia (APL) relapsing after upfront arsenic trioxide (ATO) therapy is not defined. In this phase II study we demonstrate the safety of adding a proteasome inhibitor with ATO in the management of relapsed APL treated with upfront ATO. We also demonstrate the efficacy of this combination and the potential for its utilization in this setting.

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it's efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34 sorted cells when exposed to ATO lead to a significant decrease in the expression of , ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33-46) vs 54 days (range: 52-75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.