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.

Modulating retinoid-X-receptor alpha (RXRA) expression sensitizes chronic myeloid leukemia cells to imatinib in vitro and reduces disease burden in vivo.

Introduction: The ligand-activated transcription factors, nuclear hormone receptors (NHRs), remain unexplored in hematological malignancies except for retinoic acid receptor alpha (RARA). Methods: Here we profiled the expression of various NHRs and their coregulators in Chronic myeloid leukemia (CML) cell lines and identified a significant differential expression pattern between inherently imatinib mesylate (IM)-sensitive and resistant cell lines. Results: Retinoid-X-receptor alpha (RXRA) was downregulated in CML cell lines inherently resistant to IM and in primary CML CD34+ cells. Pre-treatment with clinically relevant RXRA ligands improved sensitivity to IM in-vitro in both CML cell lines and primary CML cells. This combination effectively reduced the viability and colony-forming capacity of CML CD34+ cells in-vitro. In-vivo, this combination reduced leukemic burden and prolonged survival. Overexpression (OE) of RXRA inhibited proliferation and improved sensitivity to IM in-vitro. In-vivo, RXRA OE cells showed reduced engraftment of cells in the bone marrow, improved sensitivity to IM, and prolonged survival. Both RXRA OE and ligand treatment markedly reduced BCR::ABL1 downstream kinase activation, activating apoptotic cascades and improving sensitivity to IM. Importantly, RXRA OE also led to the disruption of the oxidative capacity of these cells. Conclusion: Combining IM with clinically available RXRA ligands could form an alternative treatment strategy in CML patients with suboptimal response to IM.

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

NUDT15 c.415C>T Polymorphism Predicts 6-MP Induced Early Myelotoxicity in Patients with Acute Lymphoblastic Leukemia Undergoing Maintenance Therapy.

PURPOSE: Severe myelosuppression in patients with acute lymphoblastic leukemia (ALL) undergoing 6-MP-based maintenance therapy is attributed to TPMT gene polymorphisms, which is rare in Asian populations. This study aims to evaluate the role of selected polymorphisms in NUDT15, ITPA, and MRP4 genes in addition to TPMT in predicting 6-MP intolerance during ALL maintenance therapy. PATIENTS AND METHODS: We screened for the presence of NUDT15*3 (c.415 C>T, rs116855232); MRP4 c.2269 C>T (rs3765534), ITPA c.94 C>A (rs1127354) polymorphisms in addition to TPMT *2 (rs1800462), *3A (*3B and *3C; rs1800460 and rs1142345) in ALL patients with documented severe neutropenia (cohort-1; n=42). These polymorphisms were then screened in a prospective cohort of ALL patients (cohort-2; n=133) and compared with 6-MP dose reduction, early/late myelotoxicity. RESULTS: Nineteen (45%) patients in cohort-1 and 18 (14%) in cohort-2 had NUDT15 c.415 C>T variant while 4 (3%) patients in cohort-2 had TPMT*3C variant. Five (12%) in cohort-1 and 30 (24%) in cohort-2 had ITPA c.94 C>A variant while 9 (22%) and 15 (12%) had MRP4 c.2269 C>T variant in cohorts-1 and 2, respectively. All in cohort-1 and 36 (27%) in cohort-2 had severe myelotoxicity. Twenty-eight patients (66.6%) in cohort-1 and 40 (30%) patients in cohort-2 had significant 6-MP dose reduction. NUDT15 c.415 C>T variant explained severe myelotoxicity in 63% and 33% in cohort 1 and 2. TPMT*3C and ITPA c.94 C>A variants also explained myelotoxicity in cohort-2 (Median ANC: 376 vs 1014 mm3; p=0.04 and 776 vs 1023 mm3; p=0.04 respectively). NUDT15 c.415 C>T polymorphism explained significant myelotoxicity (507 vs 1298 mm3; p<0.0001) in the multivariate analysis as well (ß=-0.314, p<0.0001). CONCLUSION: NUDT15 c.415 C>T (15*3), TPMT*3C, as well as ITPA c.94 C>A and MRP4 c.2269 C>T polymorphisms explain hematotoxicities. Preemptive genotype-based (NUDT15*3, TPMT, ITPA c.94 C>A) 6-MP dosing could improve the outcome after maintenance therapy.

Plasma imatinib levels and ABCB1 polymorphism influences early molecular response and failure-free survival in newly diagnosed chronic phase CML patients.

Achieving early molecular response (EMR) has been shown to be associated with better event free survival in patients with chronic phase chronic myeloid leukemia (CP-CML) on Imatinib therapy. We prospectively evaluated the factors influencing the 2-year failure free survival (FFS) and EMR to imatinib therapy in these patients including day29 plasma Imatinib levels, genetic variants and the gene expression of target genes in imatinib transport and biotransformation. Patients with low and intermediate Sokal score had better 2-year FFS compared to those with high Sokal Score (p = 0.02). Patients carrying ABCB1-C1236T variants had high day29 plasma imatinib levels (P = 0.005), increased EMR at 3 months (P = 0.044) and a better 2 year FFS (P = 0.003) when compared to those with wild type genotype. This translates to patients with lower ABCB1 mRNA expression having a significantly higher intracellular imatinib levels (P = 0.029). Higher day29 plasma imatinib levels was found to be strongly associated with patients achieving EMR at 3 months (P = 0.022), MMR at 12 months (P = 0.041) which essentially resulted in better 2-year FFS (p = 0.05). Also, patients who achieved EMR at 3 months, 6 months and MMR at 12 months had better FFS when compared to those who did not. This study suggests the incorporation of these variables in to the imatinib dosing algorithm as predictive biomarkers of response to Imatinib therapy.