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Background Myelofibrosis is a myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferative and anomalous production of cytokines with genetic mutations in the JAK/STAT signalling pathway playing a distinctive role in its pathophysiology. Diagnosis of MF presents a challenge due to vague and overlapping symptoms. The present strategy for managing MF is not well defined and relies on a symptomatic approach. Ruxolitinib is the first drug approved by the United States Food and Drug Administration in 2011. Drug Controller General of India approved Ruxolitinib in 2013 as a first-line treatment in patients with MF. This comprehensive survey aims to understand the MF patient journey and the perceptions/practices of Indian hematologists/oncologists regarding diagnosis, prognosis, and disease management of myelofibrosis. Methods A cross-sectional, multicentric, qualitative survey was conducted across 17 Indian cities from October 2021 to November 2021. One-on-one telephonic interviews were conducted using a structured questionnaire based on the study objective. Descriptive statistics were used to analyse the obtained data. Results Overall, 50 physicians and 154 patients (primary MF: 51, post-PV: 78 and post-ET: 25) completed the survey. The most common symptoms reported by patients and physicians at the time of diagnosis were, abdominal pain/discomfort (81% vs. 70%), fatigue/tiredness/weakness (77% vs. 73%) and fever (54% vs. 48%). A 10-month delay was observed from the symptom onset to the final diagnosis and a further 9-month lag from final diagnosis to treatment initiation. In our survey, the physicians preferred Hydroxyurea (88%), blood transfusion (82%) and Ruxolitinib (78%) as the treatment regimen. The majority of the patients were aware of their treatment. Hydroxyurea constituted the predominant treatment option ( n  = 85); however, satisfaction was highest with Ruxolitinib (50%, n  = 13). The physicians considered improving overall survival as an important treatment goal, while patients prioritized symptom relief. Conclusion The survey highlights the importance of understanding symptom burden and treatment goal perceptions in shaping management decisions. The results emphasize the need to align the physicians and patients on the actual treatment outcomes through patient education. Strengthening this alignment will enhance treatment adherence, improve patient satisfaction, and ensure better clinical outcomes, ultimately leading to a more patient-centred approach to managing the disease.

Background Myelofibrosis (MF), leads to variety of symptoms, significantly impacting patients’ quality of life (QoL) and work productivity. Limited data exists on MF in India. This survey conducted among Indian physicians and their patients with MF evaluated patient journey, understand perceptions around symptom burden, its impact on QoL, and management. Methods This cross-sectional and multicentric survey between October to November 2021 across 17 cities was conducted using a structured questionnaire based on certain identified themes (symptom burden, QoL parameters, treatment goals and patient communication) during one-on-one telephonic interview. Descriptive statistics were used for data analysis. Results Fifty physicians and 154 patients (primary MF: 51, post-PV: 78, post-ET: 25) participated. Diagnostic delay of 10 months from symptom onset was noted. 151 (98%) patients reported reduced QoL and loss of work productivity. Majority of the patients (56%) reported a negative impact on work productivity, with 76% confirming that MF hindered their work-related daily activities and 42% having reduced work hours. Key symptoms included abdominal pain (patients: 81%, physicians: 70%), fatigue (77% vs. 73%), and fever (54% vs. 48%). Patient-physician perception discrepancies on QoL factors were notable, particularly bone pain (12% vs. 64%), sleep difficulties (8% vs. 70%), sexual problems (2% vs. 78%), depression (4% vs. 63%), and inactivity (4% vs. 66%). Conclusion The study results highlight the negative impact of MF on work productivity by hampering their work-related daily activities and reducing the work hours. The findings underscore the significance of enhancing patient-physician communication and standardizing monitoring practices for improving care outcomes.

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR- mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs. Lineage tracing analyses of cells from two monozygotic twins presenting with myelofibrosis in adulthood provide evidence of in utero transplacental transmission of the tumorigenic clone.

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.

Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.

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

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.

The persistence of CAR T cells and antigen escape remain major barriers to durable therapeutic success in hematologic malignancies. Our study integrates AI-guided design with targeted protein degradation to overcome these challenges. Utilizing an in-silico library of CAR constructs followed by an in vitro screening, we developed a predictive model, CARMSeD, which forecasts constructs prone to self-activation and dysfunction. Optimized bispecific CD20/CD19 CAR T cells demonstrate superior persistence and anti-tumor efficacy. To further improve durability, the platform incorporates a PROTAC-based module that selectively degrades AKT3, promoting FOXO4-driven mitochondrial fitness, central memory differentiation, and reduced mTOR signaling. We extended this strategy to develop a trispecific CAR T platform co-expressing a secretable CD3/CD22 bispecific engager, achieving potent tumor eradication even in CD19/CD20-negative malignancies demonstrates efficacy across patient-derived leukemia samples and solid tumor models. Together, our study introduces a next-generation AI-guided CAR T strategy that integrates structure-based optimization and intracellular modulation to improve persistence, broaden antigen coverage, and ensure durable therapeutic efficacy. Durable, multi-antigen CAR T responses in B-cell malignancies are in need. The authors here demonstrate that AI-guided CAR designs combined with targeted pathway modulation enhance persistence, prevent antigen escape, and improve anti-tumor efficacy.

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.