Metformin sensitizes AML cells to venetoclax through endoplasmic reticulum stress-CHOP pathway.

Venetoclax inhibits acute myeloid leukaemia by inhibiting BCL-2 targeting, and a combination regimen with venetoclax has been explored. Although these regimens produce better clinical results, the vast majority of patients still suffer from disease recurrence or primary drug resistance. Metformin has been demonstrated to induce apoptosis in cancer cells. However, whether it can synergize with venetoclax and the underlying mechanisms of metformin-induced apoptosis are not fully understood. In this study, we investigated the effect of metformin and venetoclax on the growth of AML cells in vitro and in vivo. In both Molm13 and THP-1 cell lines, metformin and venetoclax synergistically inhibited the proliferation and induced apoptosis of leukaemia cells. Most importantly, the combination of metformin and venetoclax treatment significantly increased the expression levels of the endoplasmic reticulum (ER) stress-related marker CHOP, for example, in AML cell lines. Knockdown of CHOP markedly attenuated the metformin- and venetoclax-induced cell apoptosis. Moreover, the combination of metformin and venetoclax demonstrated prominent anti-leukaemia effects in xenograft models and bone marrow samples from AML patients. In summary, the combination of metformin and venetoclax showed enhanced anti-leukaemia activity with acceptable safety in AML patients, representing a new combinatorial strategy worth further clinical investigation to treat AML.

Protocol for detecting lysosome quantity and membrane permeability in acute myeloid leukemia cell lines.

Lysosomal function and activity are essential to support cellular adaptation to multiple stresses. For example, certain drugs can induce increased lysosomal membrane permeability to exert their anti-cancer effects. Here, we present a protocol to evaluate the lysosome alterations induced by drug treatment. We first describe the steps for inducing lysosomal alterations in cultured cells. We then show how to quantify the number of lysosomes, assess the integrity of lysosomal membranes, and determine lysosomal membrane permeabilization by using galectin puncta assay. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.

Hexamethylene amiloride synergizes with venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia.

Tumors maintain an alkaline intracellular environment to enable rapid growth. The proton exporter NHE1 participates in maintenance of this pH gradient. However, whether targeting NHE1 could inhibit the growth of tumor cells remains unknown. Here, we report that the NHE1 inhibitor Hexamethylene amiloride (HA) efficiently suppresses the growth of AML cell lines. Moreover, HA combined with venetoclax synergized to efficiently inhibit the growth of AML cells. Interestingly, lysosomes are the main contributors to the synergism of HA and venetoclax in inhibiting AML cells. Most importantly, the combination of HA and venetoclax also had prominent anti-leukemia effects in both xenograft models and bone marrow samples from AML patients. In summary, our results provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML in vitro and in vivo.

Cxcl10 and Cxcr3 regulate self-renewal and differentiation of hematopoietic stem cells.

BACKGROUND: The function of hematopoietic stem cells (HSC) is regulated by HSC internal signaling pathways and their microenvironment. Chemokines and chemokine ligands play important roles in the regulation of HSC function. Yet, their functions in HSC are not fully understood. METHODS: We established Cxcr3 and Cxcl10 knockout mouse models (Cxcr3-/- and Cxcl10-/-) to analyze the roles of Cxcr3 or Cxcl10 in regulating HSC function. The cell cycle distribution of LT-HSC was assessed via flow cytometry. Cxcr3-/- and Cxcl10-/- stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. To study the effects of Cxcr3 or Cxcl10 deficient bone marrow microenvironment, we transplanted CD45.1 donor cells into Cxcr3-/-or Cxcl10-/- recipient mice (CD45.2) and examined donor-contributed hematopoiesis. RESULTS: Deficiency of Cxcl10 and its receptor Cxcr3 led to decreased BM cellularity in mice, with a significantly increased proportion of LT-HSC. Cxcl10-/- stem/progenitor cells showed reduced self-renewal capacity in the secondary transplantation assay. Notably, Cxcl10-/- donor-derived cells preferentially differentiated into B lymphocytes, with skewed myeloid differentiation ability. Meanwhile, Cxcr3-deficient HSCs demonstrated a reconstitution disadvantage in secondary transplantation, but the lineage bias was not significant. Interestingly, the absence of Cxcl10 or Cxcr3 in bone marrow microenvironment did not affect HSC function. CONCLUSIONS: The Cxcl10 and Cxcr3 regulate the function of HSC, including self-renewal and differentiation, adding to the understanding of the roles of chemokines in the regulation of HSC function.

PHF6 mutation is associated with poor outcome in acute myeloid leukaemia.

INTRODUCTION: Mutation of plant homeodomain finger protein 6 (PHF6) occurs in approximately 3% of acute myeloid leukaemia (AML) cases. Although it was reported to be associated with poor prognosis, it was not confirmed by other groups. Recently, propensity score matching has provided an effective way to minimise bias by creating two groups that are well balanced with respect to baseline characteristics, providing more convincing results, which has an advantage, especially for rare subtype studies. To provide further evidence on the role of PHF6 mutation, we performed a retrospective propensity score-matched cohort study to assess the therapeutic responses and survival outcomes of AML patients with PHF6 mutation compared with those without PHF6 mutation after balancing age, sex and risk categories. PATIENTS AND METHODS: A total of 22 patients with PHF6 mutation from 801 consecutive newly diagnosed AML cases in our center were identified, and 43 patients with the PHF6 wild-type genotype were successfully matched at a 1:2 ratio. RESULTS: AML harbouring PHF6 mutation was associated with a lower complete remission (CR) rate (41% vs. 69%; OR = 3.64, 95% CI 1.10, 12.10; p = 0.035) and shorter median overall survival (OS) (6.0 vs. 39.0 months; p < 0.001) and event-free survival (EFS) (2.0 vs. 11.0 months; p = 0.013) compared with PHF6 wild-type patients. Further multivariate analysis supported that PHF6 mutation was an independent risk factor for overall survival in AML (HR = 8.910, 95% CI 3.51, 22.63; p < 0.001). In addition, allogeneic haematopoietic stem cell transplantation (allo-HSCT) seemed to ameliorate the poor prognosis of AML with PHF6 mutation in this study. CONCLUSION: Our data revealed that PHF6 mutation was associated with a lower chemotherapy response and shorter survival, suggesting that PHF6 mutation is a predictor of poor prognosis in AML.

Glucose partitioning in the bone marrow micro-environment in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) cells metabolise glucose by glycolysis-based re-programming. However, how glucose uptake is partitioned between leukaemia cells and other cells of the bone marrow micro-environment is unstudied. We used a positron emission tomography (PET) tracer 18F fluorodeoxyglucose ([18F]-FDG) probe and transcriptomic analyses to detect glucose uptake by diverse cells in the bone marrow micro-environment in a MLL-AF9-induced mouse model. Leukaemia cells had the greatest glucose uptake with leukaemia stem and progenitor cells having the greatest glucose uptake. We also show the effects of anti-leukaemia drugs on leukaemia cell numbers and glucose uptake. Our data suggest targeting glucose uptake as a potential therapy strategy in AML if our observations are validated in humans with AML.