Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Inhibition of NRF2 signaling overcomes acquired resistance to arsenic trioxide in FLT3-mutated Acute Myeloid Leukemia.

De novo acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) has been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro. We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML and molecular/pharmacological inhibition of NF-E2 related factor 2 (NRF2), a master regulator of antioxidant response improved the chemosensitivity to ATO and daunorubicin even in non FLT3-ITD mutated cell lines and primary samples. We examined the effects of molecular/pharmacological suppression of NRF2 on acquired ATO resistance in the FLT3-ITD mutant AML cell line (MV4-11-ATO-R). ATO-R cells showed increased NRF2 expression, nuclear localization, and upregulation of bonafide NRF2 targets. Molecular inhibition of NRF2 in this resistant cell line improved ATO sensitivity in vitro. Digoxin treatment lowered p-AKT expression, abrogating nuclear NRF2 localization and sensitizing cells to ATO. However, digoxin and ATO did not sensitize non-ITD AML cell line THP1 with high NRF2 expression. Digoxin decreased leukemic burden and prolonged survival in MV4-11 ATO-R xenograft mice. We establish that altering NRF2 expression may reverse acquired ATO resistance in FLT3-ITD AML.

Molecular characterization of triple-negative myeloproliferative neoplasms by next-generation sequencing.

The role of next-generation sequencing (NGS) in identifying mutations in the driver, epigenetic regulator, RNA splicing, and signaling pathway genes in myeloproliferative neoplasms (MPNs) has contributed substantially to our understanding of the disease pathogenesis as well as disease evolution. NGS aids in determining the clonal nature of the disease in a subset of these disorders where mutations in the driver genes are not detected. There is a paucity of real-world data on the utility of this test in the characterization of triple-negative myeloproliferative neoplasms (TN-MPN). In this study, 46 samples of TN-MPN (essential thrombocythemia (ET) = 17; primary myelofibrosis (PMF) = 23; & myeloproliferative neoplasm unclassified (MPN-u) = 6) were screened for markers of clonality using targeted NGS. Among these, 25 (54.3%) patients had mutations that would help determine the clonal nature of the disease. Eight of the 17 TN-ET (47%) and 13 of the 23 TN-PMF (56.5%) patients had noncanonical mutations in the driver genes and mutations in the genes involved in epigenetic regulation. Identification of mutations categorized as high molecular markers (HMR) in 2 patients helped classify them as PMF with high risk according to the MIPSS 70 scoring system. A novel mutation in the MPIG6B (C6orf25) gene associated with childhood myelofibrosis was detected in a 14-year-old girl. The presence of clonal hematopoiesis could be confirmed in four of the six MPN-u patients in this cohort. This study demonstrates the utility of NGS in improving the characterization of TN-MPN by establishing clonality and detecting noncanonical mutations in driver genes, thereby aiding in clinical decision-making.

Management of relapse in acute promyelocytic leukaemia treated with up-front arsenic trioxide-based regimens.

The standard of care for patients with acute promyelocytic leukaemia (APL) relapsing after front-line treatment with arsenic trioxide (ATO)-based regimens remains to be defined. A total of 67 patients who relapsed after receiving ATO-based up-front therapy and were also salvaged using an ATO-based regimen were evaluated. The median (range) age of patients was 28 (4-54) years. While 63/67 (94%) achieved a second molecular remission (MR) after salvage therapy, three (4·5%) died during salvage therapy. An autologous stem cell transplant (auto-SCT) was offered to all patients who achieved MR, 35/63 (55·6%) opted for auto-SCT the rest were administered an ATO + all-trans retinoic acid maintenance regimen. The mean (SD) 5-year Kaplan-Meier estimate of overall survival and event-free survival of those who received auto-SCT versus those who did not was 90·3 (5·3)% versus 58·6 (10·4)% (P = 0·004), and 87·1 (6·0)% versus 47·7 (10·3)% (P = 0·001) respectively. On multivariate analysis, failure to consolidate MR with an auto-SCT was associated with a significantly increased risk of relapse [hazard ratio (HR) 4·91, 95% confidence interval (CI) 1·56-15·41; P = 0·006]. MR induction with ATO-based regimens followed by an auto-SCT in children and young adults with relapsed APL who were treated with front-line ATO-based regimens was associated with excellent long-term survival.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Resource utilization and cost effectiveness of treating acute promyelocytic leukaemia using generic arsenic trioxide.

Arsenic trioxide (ATO)-based regimens are the standard of care for treating acute promyelocytic leukaemia (APL) and have replaced chemotherapy-based approaches. However, the cost of "patented" ATO is prohibitive because of patent rights. "Generic" ATO has been used in a few countries, but its implications for health resource utilization (HRU) and cost of treatment are unknown. We hypothesized that treating APL patients using generic ATO (APL-ATO) will be cost effective compared to the chemotherapy-based regimen (APL-CT). In a single-centre retrospective study, we used a bottom-up costing method to compare the direct medical cost of treatment and HRU between APL-ATO and APL-CT. These costs and the survival and relapse probabilities were imputed in a three-state Markov decision model to estimate the cost effectiveness of APL-ATO compared to APL-CT. The mean cost of treatment for APL-ATO (n = 30, $8500 ± 2078) was significantly less than for APL-CT (n = 30, $22 600 ± 5528) (P < 0·001). APL-ATO reduced hospitalization, antibiotic and antifungal usage (P < 0·001). In the Markov model, five-year treatment costs were significantly lower for APL-ATO ($11 131) than for APL-CT ($17 926) (P < 0·001). Treatment cost and health resource utilization were significantly lower for generic ATO-treated APL patients compared to the chemotherapy-based regimen.

JAK2 exon 12 mutations in cases with JAK2V617F-negative polycythemia vera and primary myelofibrosis.

Molecular detection of JAK2 mutation (V617F or exon 12) is included as a major diagnostic criterion for polycythemia vera (PV) by the WHO 2016 guidelines. JAK2 exon 12 mutations are seen in about 2-5% of JAK2V617F-negative cases of PV. Mutations in JAK2 cause constitutive activation of JAK-STAT pathway which results in variable phenotypes. PV patients with exon 12 mutations in JAK2 present characteristically with erythrocytosis. There are limited reports describing the spectrum of JAK2 exon12 mutations in myeloproliferative neoplasms (MPNs). Here, we describe the characteristics of a series of MPN patients with mutations in exon 12 of JAK2 of which two were novel variants associated with polycythemia. Interestingly, we noted two patients presenting as myelofibrosis having JAK2 exon 12 mutations.

Acute myeloid leukaemia: challenges and real world data from India.

The management of acute myeloid leukaemia (AML) in India remains a challenge. In a two-year prospective study at our centre there were 380 newly diagnosed AML (excluding acute promyelocytic leukaemia, AML-M3) patients. The median age of newly diagnosed patients was 40 years (range: 1-79; 12.3% were ≤ 15 years, 16.3% were ≥ 60 years old) and there were 244 (64.2%) males. The median duration of symptoms prior to first presentation at our hospital was 4 weeks (range: 1-52). The median distance from home to hospital was 580 km (range: 6-3200 km). 109 (29%) opted for standard of care and were admitted for induction chemotherapy. Of the 271 that did not take treatment the major reason was lack of financial resources in 219 (81%). There were 27 (24.7%) inductions deaths and of these, 12 (44.5%) were due to multidrug-resistant gram-negative bacilli and 12 (44.5%) showed evidence of a fungal infection. The overall survival at 1 year was 70.4% ± 10.7%, 55.6% ± 6.8% and 42.4% ± 15.6% in patients aged ≤ 15 years, 15 - 60 years and ≥ 60 years, respectively. In conclusion, the biggest constraint is the cost of treatment and the absence of a health security net to treat all patients with this diagnosis.

Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy.

Data on minimal residual disease (MRD) monitoring in acute promyelocytic leukemia (APL) are available only in the context of conventional all-trans retinoic acid plus chemotherapy regimens. It is recognized that the kinetics of leukemia clearance is different with the use of arsenic trioxide (ATO) in the treatment of APL. We undertook a prospective peripheral blood RT-PCR-based MRD monitoring study on patients with APL treated with a single agent ATO regimen. A total of 151 patients were enrolled in this study. A positive RT-PCR reading at the end of induction therapy was significantly associated on a multivariate analysis with an increased risk of relapse (relative risk = 4.9; P = .034). None of the good risk patients who were RT-PCR negative at the end of induction relapsed. The majority of the relapses (91%) happened within 3 years of completion of treatment. After achievement of molecular remission, the current MRD monitoring strategy was able to predict relapse in 60% of cases with an overall sensitivity and specificity of 60% and 93.2%, respectively. High-risk group patients and those that remain RT-PCR positive at the end of induction are likely to benefit from serial MRD monitoring by RT-PCR for a period of 3 years from completion of therapy.

Immortalised chronic myeloid leukemia (CML) derived mesenchymal stromal cells (MSCs) line retains the immunomodulatory and chemoprotective properties of CML patient-derived MSCs.

Despite the success of Tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML), leukemic stem cells (LSCs) persist, contributing to relapse and resistance. CML Mesenchymal Stromal Cells (MSCs) help in LSC maintenance and protection from TKIs. However, the limited passage and self-differentiation abilities of primary CML MSCs hinder extensive research. To overcome this, we generated and characterized an immortalised CML patient-derived MSC (iCML MSC) line and assessed its role in LSC maintenance. We also compared the immunophenotype and differentiation potential between primary CML MSCs at diagnosis, post-treatment, and with normal bone marrow MSCs. Notably, CML MSCs exhibited enhanced chondrogenic differentiation potential compared to normal MSCs. The iCML MSC line retained the trilineage differentiation potential and was genetically stable, enabling long-term investigations. Functional studies demonstrated that iCML MSCs protected CML CD34+ cells from imatinib-induced apoptosis, recapitulating the bone marrow microenvironment-mediated resistance observed in patients. iCML MSC-conditioned media enabled CML CD34+ and AML blast cells to proliferate rapidly, with no impact on healthy donor CD34+ cells. Gene expression profiling revealed dysregulated genes associated with calcium metabolism in CML CD34+ cells cocultured with iCML MSCs, providing insights into potential therapeutic targets. Further, cytokine profiling revealed that the primary CML MSC lines abundantly secreted 25 cytokines involved in immune regulation, supporting the hypothesis that CML MSCs create an immune modulatory microenvironment that promotes growth and protects against TKIs. Our study establishes the utility of iCML MSCs as a valuable model to investigate leukemic-stromal interactions and study candidate genes involved in mediating TKI resistance in CML LSCs.

The Outcome of Pediatric Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Experience from a Referral Center in South India.

Although improved survival in children with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) has been demonstrated in trials, the outcome appears to be inferior in low- and middle-income countries (LMIC). Methods A file review of children aged ≤ 15 years diagnosed with Ph-ALL from 2010 to 2019 was performed. Minimal residual disease (MRD) was assessed by flow-cytometry. Real-time polymerase chain reaction (qRT-PCR) was used to quantify the BCR::ABL1 transcripts during treatment. Results The mean age of the 20 patients in the study was 91 months. Of 19 patients in whom the BCR::ABL1 transcript was confirmed, 10(50%) had P210, 7(35%) had P190, and two showed dual expression. The mean dose of imatinib that was administered was 294 ± 41 mg/m2/day. qRT-PCR for BCR::ABL1 was < 0.01% in all patients who were in remission or had a late relapse and was ≥ 0.01% in patients who had an early relapse. Two patients underwent HSCT. The 3-year event-free survival (EFS) was 35.0 ± 10.7%. Patients with a good prednisolone response (GPR) and a negative end-of-induction MRD demonstrated a superior EFS to those who lacked either or both (80.0 ± 17.9% vs. 16.7 ± 15.2%, P = 0.034). Conclusion The 3-year EFS of 20 children with Ph-ALL treated with chemotherapy and TKI was < 50%. An unusually high proportion of patients with p210 transcript expression; sub-optimal TKI dosing and lesser intensity of chemotherapy, due to the concern of high treatment-related mortality in LMIC are possible reasons for the poor outcome. Conventional treatment response parameters such as GPR and MRD predict outcomes in Ph-ALL. qRT-PCR for BCR::ABL1 may have a role in predicting early relapse. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01684-9.

Treosulfan Exposure Predicts Thalassemia-Free Survival in Patients with Beta Thalassemia Major Undergoing Allogeneic Hematopoietic Cell Transplantation.

A toxicity-reduced conditioning regimen with treosulfan, fludarabine, and thiotepa in patients with high-risk ß-thalassemia major has significantly improved hematopoietic stem cell transplantation (HCT) outcomes. However, complications resulting from regimen-related toxicities (RRTs), mixed chimerism, and graft rejection remain a challenge. We evaluated the dose-exposure-response relationship of treosulfan and its active metabolite S, S-EBDM, in a uniform cohort of patients with ß-thalassemia major to identify whether therapeutic drug monitoring (TDM) and dose adjustment of treosulfan is feasible. Plasma treosulfan/S, S-EBDM levels were measured in 77 patients using a validated liquid chromatography with tandem mass spectrometry method, and the pharmacokinetic parameters were estimated using nlmixr2. The influence of treosulfan and S, S-EBDM exposure, and GSTA1/NQO1 polymorphisms on graft rejection, RRTs, chimerism status, and 1-year overall survival (OS), and thalassemia-free survival (TFS) were assessed. We observed that treosulfan exposure was lower in patients with graft rejection than those without (1,655 vs. 2,037 mg•h/L, P = 0.07). Pharmacodynamic modeling analysis to identify therapeutic cutoff revealed that treosulfan exposure ≥1,660 mg•hour/L was significantly associated with better 1-year TFS (97% vs. 81%, P = 0.02) and a trend to better 1-year OS (90% vs. 69%, P = 0.07). Further, multivariate analysis adjusting for known pre-HCT risk factors also revealed treosulfan exposure <1,660 mg•h/L (hazard ratio (HR) = 3.23; 95% confidence interval (CI) = 1.12-9.34; P = 0.03) and GSTA1*B variant genotype (HR = 3.75; 95% CI = 1.04-13.47; P = 0.04) to be independent predictors for inferior 1-year TFS. We conclude that lower treosulfan exposure increases the risk of graft rejection and early transplant-related mortality affecting TFS. As no RRTs were observed with increasing treosulfan exposure, TDM-based dose adjustment could be feasible and beneficial.

Precise correction of a spectrum of ß-thalassemia mutations in coding and non-coding regions by base editors.

ß-thalassemia/HbE results from mutations in the ß-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of ß-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of ß-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with ß-thalassemia/HbE. These ß-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional ß-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe ß0/ßE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

A rapid & sensitive liquid chromatography- tandem mass spectrometry method for the quantitation of busulfan levels in plasma & application for routine therapeutic monitoring in haematopoietic stem cell transplantation.

BACKGROUND & OBJECTIVES: Busulfan (Bu) in combination with cyclophosphamide is widely used in myeloablative conditioning regimen prior to haematopoietic stem cell transplantation (HSCT). Its narrow therapeutic range and toxic side effects at high systemic exposure and graft rejection at low exposure emphasize the need for busulfan dose optimization using targeted dose adjustment prior to HSCT. We report here a rapid and sensitive method to quantitate busulfan plasma levels in patients receiving busulfan as part of pre-transplant conditioning. METHODS: The method involves simple protein precipitation of the plasma followed by analysis using a high performance liquid chromatography (HPLC) with tandem mass spectrometry - electrospray ionization technique (LC-ESI MS/MS) in positive ionization mode and quantified using multiple reaction monitoring (MRM). Deuterated busulfan (d8-busulf`an) was used as the internal standard. RESULTS: The method was linear for the concentration ranging from 0 to 4000 ng/ml of busulfan with a limit of detection of 2 ng/ml and limit of quantitation of 5 ng/ml. The assay was accurate for serial concentrations of Bu in plasma for five consecutive days and the CV was less than 10 per cent. CONCLUSION: Using this rapid and sensitive method, busulfan levels were targeted and subsequent doses adjusted at our center in 26 patients receiving high dose busulfan in combination with cyclophosphamide or fludarabine.

Biomarkers for early complications post hematopoietic cell transplantation: Insights and challenges.

Hematopoietic cell transplantation is an established curative treatment option for various hematological malignant, and non-malignant diseases. However, the success of HCT is still limited by life-threatening early complications post-HCT, such as Graft Versus Host Disease (GVHD), Sinusoidal Obstruction Syndrome (SOS), and transplant-associated microangiopathy, to name a few. A decade of research in the discovery and validation of novel blood-based biomarkers aims to manage these early complications by using them for diagnosis or prognosis. Advances in this field have also led to predictive biomarkers to identify patients' likelihood of response to therapy. Although biomarkers have been extensively evaluated for different complications, these are yet to be used in routine clinical practice. This review provides a detailed summary of various biomarkers for individual early complications post-HCT, their discovery, validation, ongoing clinical trials, and their limitations. Furthermore, this review also provides insights into the biology of biomarkers and the challenge of obtaining a universal cut-off value for biomarkers.

Metformin pretreatment ameliorates busulfan-induced liver endothelial toxicity during haematopoietic stem cell transplantation.

The success of Haematopoietic cell transplantation (HCT) is often limited by regimen-related toxicity (RRT) caused by conditioning regimen drugs. Among different conditioning drugs, busulfan (Bu) and treosulfan (Treo), although widely used in HCT, exhibit different toxicity profiles, the mechanism of which is still unclear. Here we investigated the effects of Bu and Treo in endothelial cells. While both Bu and Treo induced DNA damage in endothelial cells, we observed Bu alone to induce oxidative stress and sustained activation of phospho-ERK1/2, leading to apoptosis. However, Treo-treated cells exhibited no oxidative stress/apoptosis of endothelial cells. Screening of pharmacological inhibitors of both ROS and p-ERK revealed that metformin effectively ameliorates Bu-mediated toxicity in endothelial cells. In Balb/c mice, we observed a significant reduction in bone marrow endothelial cells in Bu-treated mice compared to Treo-treated mice. Further, liver sinusoidal endothelial cells (LSEC) was damaged by Bu, which is implicated in liver vasculature and their functional capacity to uptake FITC-albumin. However, Treo-treated mice liver vasculature was morphologically and functionally normal. When mice were pretreated with metformin followed by Bu, LSECs damage was ameliorated morphologically and functionally. Bone marrow transplants done on these mice did not affect the engraftment of donor cells.

Clinical significance of end of induction measurable residual disease monitoring in B-cell acute lymphoblastic leukemia: A single center experience.

The assessment of measurable residual disease (MRD) has emerged as a powerful prognostic tool for both pediatric and adult acute lymphoblastic leukemia (ALL). This retrospective study aimed to evaluate the prognostic relevance of the end of induction MRD in B-cell acute lymphoblastic leukemia (B ALL) patients. The study included 481 patients who underwent treatment for B ALL between August 2012 and March 2019 and had their MRD at the end of induction assessed by flow cytometry. Baseline demographic characteristics were collected from the patient's clinical records. Event free survival (EFS) and relapse free survival (RFS) were calculated using Kaplan-Meier analysis and survival estimates were compared using the log-rank test. End of induction MRD and baseline karyotype were the strongest predictors of EFS and RFS on multivariate analysis. The EFS was inversely related to the MRD value and the outcomes were similar in patients without morphological remission at the end of induction and patients in remission with MRD ≥1.0%. Even within the subgroups of ALL based on age, karyotype, BCR::ABL1 translocation and the treatment protocol, end of induction MRD positive patients had poor outcomes compared to patients who were MRD negative. The study outcome would help draft end of induction MRD-based treatment guidelines for the management of B ALL patients.

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.

Chemotherapeutic drugs elicit stemness and metabolic alteration to mediate acquired drug-resistant phenotype in acute myeloid leukemia cell lines.

Chemotherapy resistance leading to disease relapse is a significant barrier in treating acute myeloid leukemia (AML). Metabolic adaptations have been shown to contribute to therapy resistance. However, little is known about whether specific therapies cause specific metabolic changes. We established cytarabine-resistant (AraC-R) and Arsenic trioxide-resistant (ATO-R) AML cell lines, displaying distinct cell surface expression and cytogenetic abnormalities. Transcriptomic analysis revealed a significant difference in the expression profiles of ATO-R and AraC-R cells. Geneset enrichment analysis showed AraC-R cells rely on OXPHOS, while ATO-R cells on glycolysis. ATO-R cells were also enriched for stemness gene signatures, whereas AraC-R cells were not. The mito stress and glycolytic stress tests confirmed these findings. The distinct metabolic adaptation of AraC-R cells increased sensitivity to the OXPHOS inhibitor venetoclax. Cytarabine resistance was circumvented in AraC-R cells by combining Ven and AraC. In vivo, ATO-R cells showed increased repopulating potential, leading to aggressive leukemia compared to the parental and AraC-R. Overall, our study shows that different therapies can cause different metabolic changes and that these metabolic dependencies can be used to target chemotherapy-resistant AML.