Ablation of Wnt signaling in bone marrow stromal cells overcomes microenvironment-mediated drug resistance in acute myeloid leukemia.

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.

Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia.

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.

Decentralized manufacturing of anti CD19 CAR-T cells using CliniMACS Prodigy®: real-world experience and cost analysis in India.

Chimeric Antigen Receptor (CAR) T cell therapy is an accepted standard of care for relapsed/refractory B cell malignancies. However, the high cost of existing industry-driven centralized production makes this therapy unaffordable in low and middle-income countries. Decentralized or point of care manufacturing has the potential to overcome some of these challenges. Here we demonstrate a decentralized manufacturing process for anti-CD19-CAR-T cells using a fully automated closed system (Miltenyi CliniMACS Prodigy®) is feasible in a developing country setting. Validation run data, as part of a pre-clinical trial safety evaluation, demonstrates the successful and robust manufacturing of anti-CD19 CAR-T cells with T cell expansion of 25 to 47-fold. The median transduction efficiency was 48.8%, with a median viability of 98% and fulfillment of all standard release criteria assays for clinical application. Evaluation of production costs in an academic, not for profit setting in India provide a benchmark for low and middle-income pricing which could greatly increase access to this therapy. Based on our analysis, the cost per product would be approximately $35,107 US dollars. Our data highlights the safety, efficacy, and reproducibility of the process for use in planned future clinical trials.

Generation of good manufacturing practice grade virus-specific T cells for the management of post-transplant CMV infections.

Adoptive transfer of antigen-specific T cells has recently emerged as a successful strategy to treat viral infections following hematopoietic cell transplantation (HCT). Ex-vivo expanded donor-derived virus-specific T cells (VSTs) can be safe and effective, devoid of all the drug-related adverse effects. The study aimed to manufacture cGMP grade VSTs from healthy donors, characterize the VST product and demonstrate its safety and efficacy. Peripheral blood mononuclear cells (PBMCs) collected from six healthy donors were stimulated with pepmix that mimics the pp65 antigenic epitope of CMV and cultured for 14 days in G-Rex culture tubes. Post pepmix exposure and expansion the median CD3% was 98.8% (range:95.5% to 99.9%) while the median CD4% and CD8% were 49.1% (range:21.3% to 86.6%) and 43.9% (range:12.7% to 75.5%) respectively. The percentage of IFNγ+ cells was much higher among the CD8+ T cells (median - 18.47%; range 6.50% - 45.82%) when compared to CD4+ T cells (median - 2.74%; range 0.47% - 18.58%) and there was a switch from the CD45RA+ naive phenotype to CD45RA- effector memory phenotype in the 4 samples that achieved a >5 fold expansion. The VSTs were cytotoxic to the pepmix pulsed lymphoblasts (efficacy) while they did not induce cytolysis in the lymphoblasts that were not exposed to the pepmix (safety). This feasibility exercise helped us optimize the starting cell dose for the culture and clinical grade culture strategies, subset characterization and cytotoxicity assays. The approach could be applied to the clinical practice where virus-specific T cell infusions could be given for post-transplant viral infections.

Metabolic adaptation drives arsenic trioxide resistance in acute promyelocytic leukemia.

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are less sensitive to all-trans retinoic acid (ATRA) and the combination of ATO and ATRA compared with the sensitive cell line. Characterization of these resistant cell lines that were generated in-house showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO-resistant APL cell lines. Glycolytic inhibition by 2-deoxyglucose (2-DG) was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO-resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that combining mitocans with ATO can overcome ATO resistance. We also show that this combination has potential for treating non-M3 acute myeloid leukemia (AML) and relapsed APL. The translation of this approach in the clinic needs to be explored further.

Combination Lenalidomide/Bortezomib Treatment Synergistically Induces Calpain-Dependent Ikaros Cleavage and Apoptosis in Myeloma Cells.

Multiple myeloma had been successfully treated by combining lenalidomide and bortezomib with reports suggesting benefits of such a combination even in relapsed/refractory cases. Recently, it was demonstrated that Ikaros degradation by lenalidomide happens via proteasome-dependent pathway and this process is critical for the eradication of myeloma cells. On the basis of this, an antagonistic effect should be observed if a combination of both these agents were used, which however is not the observation seen in the clinical setting. Our study demonstrates that when these agents are combined they exhibit a synergistic activity against myeloma cells and degradation of Ikaros happens by a proteasome-independent calcium-induced calpain pathway. Our study identifies the crucial role of calcium-induced calpain pathway in inducing apoptosis of myeloma cells when this combination or lenalidomide and bortezomib is used. We also report that this combination enhanced the expression of CD38 compared with lenalidomide alone. Thus, data from our study would establish the rationale for the addition of daratumumab along with this combination to further enhance therapeutic activity against multiple myeloma. IMPLICATIONS: Lenalidomide and bortezomib combination degrades IKZF1 in multiple myeloma through a calcium-dependent calpain and caspase pathway. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/4/529/F1.large.jpg.

Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia.

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.

Prevalence of FVIII inhibitors in severe haemophilia A patients: Effect of treatment and genetic factors in an Indian population.

INTRODUCTION: Factor replacement therapy in treatment of haemophilia A is complicated by the production of neutralising antibodies known as inhibitors. The formation of inhibitors is multifactorial being associated with both genetic and environmental factors. AIM: To document the prevalence of inhibitors in severe haemophilia in the community where most patients receive only infrequent episodic replacement therapy and evaluate the factors which could be contributing to it. METHODS: Community based camps were conducted in different parts of the country. Patients were assessed through a structured questionnaire and blood samples were obtained for laboratory evaluation of inhibitors and defined immunological parameters. RESULTS: Inhibitors were present in 87/447 (19.5%) of the evaluated patients. High-titre inhibitor (>5 Bethesda Units [BU]) was identified in 31 (35.6%) patients. HLA DRB1-13-positive cases (RR = 2.04; 95% CI 1.06-3.911; P = 0.033) had an increased risk of inhibitor formation which was retained in the high-titre subset. A decreased risk of inhibitor formation was noted with heterozygous IL4-590 C/T allele (RR = 0.22; 95% CI 0.108-0.442: P = 0.000). There were no significant correlations between any of the evaluated environmental factors and the development of inhibitors in this study. CONCLUSION: The overall prevalence of inhibitors in patients with severe haemophilia A is similar to that reported among patients receiving regular replacement therapy. The data from this study, limited by its retrospective and cross-sectional study design, would suggest that genetic rather than environmental are more likely to impact the development of inhibitors.

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis.

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). In vitro 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 IKZF2, 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.