Mutation analysis of BCR-ABL1 kinase domain in chronic myeloid leukemia patients with tyrosine kinase inhibitors resistance: a Malaysian cohort study.

OBJECTIVE: Mutational analysis of BCR::ABL1 kinase domain (KD) is a crucial component of clinical decision algorithms for chronic myeloid leukemia (CML) patients with failure or warning responses to tyrosine kinase inhibitor (TKI) therapy. This study aimed to detect BCR::ABL1 KD mutations in CML patients with treatment resistance and assess the concordance between NGS (next generation sequencing) and Sanger sequencing (SS) in detecting these mutations. RESULTS: In total, 12 different BCR::ABL1 KD mutations were identified by SS in 22.6% (19/84) of patients who were resistant to TKI treatment. Interestingly, NGS analysis of the same patient group revealed an additional four different BCR::ABL1 KD mutations in 27.4% (23/84) of patients. These mutations are M244V, A344V, E355A, and E459K with variant read frequency below 15%. No mutation was detected in 18 patients with optimal response to TKI therapy. Resistance to TKIs is associated with the acquisition of additional mutations in BCR::ABL1 KD after treatment with TKIs. Additionally, the use of NGS is advised for accurately determining the mutation status of BCR::ABL1 KD, particularly in cases where the allele frequency is low, and for identifying mutations across multiple exons simultaneously. Therefore, the utilization of NGS as a diagnostic platform for this test is very promising to guide therapeutic decision-making.

Chronic myeloid leukaemia: Biology and therapy.

Chronic myeloid leukaemia (CML) is caused by BCR::ABL1. Tyrosine kinase-inhibitors (TKIs) are the initial therapy. Several organizations have reported milestones to evaluate response to initial TKI-therapy and suggest when a change of TKI should be considered. Achieving treatment-free remission (TFR) is increasingly recognized as the optimal therapy goal. Which TKI is the best initial therapy for which persons and what depth and duration of molecular remission is needed to achieve TFR are controversial. In this review we discuss these issues and suggest future research directions.

Ponatinib and STAT5 Inhibitor Pimozide Combined Synergistic Treatment Applications Potentially Overcome Drug Resistance via Regulating the Cytokine Expressional Network in Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro. The results showed that expression of interleukin (IL) 1B, IL9, and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3, IL8, IL9, IL10, IL12, or IL15. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2, IL6, IL15, IFNG, and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.

Acute Lymphoblastic Leukemia With Near-haploid Karyotype and Philadelphia Chromosome.

BACKGROUND/AIM: In precursor B-cell lineage acute lymphoblastic leukemia (BCP-ALL), leukemic cells harbor genetic abnormalities that play an important role in the diagnosis, prognosis, and treatment. A subgroup of BCP-ALL is characterized by the presence of a Philadelphia (Ph) chromosome and a chimeric BCR::ABL1 gene, whereas in another subgroup, leukemic cells exhibit near-haploidy with chromosome number 24-30. This study presents the third documented case of BCP-ALL in which a near haploid clone concurrently displayed a Ph chromosome/BCR::ABL1. CASE REPORT: Bone marrow cells obtained at diagnosis from a 25-year-old man with BCP-ALL were genetically investigated using G-banding, fluorescence in situ hybridization, and array comparative genomic hybridization. Leukemic cells had an abnormal karyotype 28,X,-Y,+6,+10,+18,+21,+ der(22) t(9;22)(q34;q11)[13]/28,idem, del(10)(q24),der(12) t(1;12) (q21;p13)[2]/46,XY[3], retained heterozygosity of the disomic chromosomes 6, 10, 18, and 21, had breakpoints in introns 1 of ABL1 and BCR, and carried a BCR::ABL1 chimera encoding the 190 kDa BCR::ABL1 protein. CONCLUSION: The coexistence of the BCR::ABL1 chimera and near-haploidy in the same cytogenetic clone suggested a possible synergistic role in leukemogenesis, with the former activating signaling pathways and the latter disrupting gene dosage balance.

The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation.

Chronic myeloid leukemia (CML) is a type of leukemia whose main genetic marker is the reciprocal translocation that leads to the production of the BCR::ABL1 oncoprotein. The expression of some genes may interfere with the progression and development of leukemias. MicroRNAs are small non-coding RNAs that have the potential to alter the expression of some genes and may be correlated with some types of leukemia and could be used as biomarkers in the diagnosis and prognosis of patients. Therefore, this project carried out an analysis of microRNA-type plasma biomarkers in patients with chronic myeloid leukemia at unique points, including follow-up analysis of patients from the Erasto Gaertner Hospital. 35 microRNAs were analyzed in different cohorts. Inside those groups, 70 samples were analyzed at unique points and 11 patients in a follow-up analysis. Statistically different results were found for microRNA-7-5p, which was found to be upregulated in patients with high expression of the BCR::ABL1 transcript when compared to healthy controls. This microRNA also had evidence of behavior related to BCR::ABL1 when analyzed in follow-up, but strong evidence was not found. In this way, this work obtained results that may lead to manifestations of a relationship between miR-7-5p and chronic myeloid leukemia, and evaluations of possible microRNAs that are not related to this pathology.

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

BACKGROUND: Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS: The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS: Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION: Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Spotlight on the real-world treatment of CML pts in Germany: a retrospective survey in private oncology practices.

Clinical trials in chronic myeloid leukemia (CML) are usually carried out in specialized centers whereas primary care for patients (pts) with CML is mainly provided by local oncology practices. The aim of this study was to assess treatment practices in pts with CML in the setting of private oncology practices in Germany. We collected data of 819 pts with a confirmed diagnosis (dx) of CML in 2013 or later from 43 practices. At dx, 84.2% (n=690) and 9.4% (n=77) of pts were in chronic or accelerated phase, 0.7% (n=6) had a blast crisis. Molecular monitoring was provided by EUTOS certified laboratories in 87.7% of pts. Typical BCR::ABL1 transcripts were detected in 86.6% (n=709). Molecular response was assessed after 2.8, 6.0, 9.4 and 12.9 m (mean) after start of treatment. Of the pts with available data, 11.1% did not achieve early molecular response and at 18 m, 83.7% had at least a major molecular response. 288 (35.2%) of pts switched to 2nd line (2L) treatment after a mean of 21.0 months. Reasons for 2L treatment were side effects in 43.4% and suboptimal response or failure in 31.4% of pts. 106 pts went on to third line (3L) treatment. 36.8 % of pts switched to and 92.8 % of pts still on 3L treatment achieved BCR::ABL1IS ≤1% at 12 m. In conclusion, in Germany pts with CML are routinely monitored by qPCR and good responses are achieved in the majority. Treatment changes are mainly due to adverse events rather than suboptimal responses.

ERG and c-MYC regulate a critical gene network in BCR::ABL1-driven B cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC-dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC-dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.

Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells.

BCR-ABL tyrosine kinase inhibitors are commonly employed for the treatment of chronic myeloid leukemia, yet their impact on human malignant melanoma remains uncertain. In this study, we delved into the underlying mechanisms of specific BCR-ABL tyrosine kinase inhibitors (imatinib, nilotinib, ZM-306416, and AT-9283) in human melanoma A375P cells. We first evaluated the influence of these inhibitors on cell growth using cell proliferation and wound-healing assays. Subsequently, we scrutinized cell cycle regulation in drug-treated A375P cells using flow cytometry and Western blot assays. Notably, imatinib, nilotinib, ZM-306416, and AT-9283 significantly reduced cell proliferation and migration in A375P cells. In particular, nilotinib and AT-9283 impeded the G1/S transition of the cell cycle by down-regulating cell cycle-associated proteins, including cyclin E, cyclin A, and CDK2. Moreover, these inhibitors reduced RB phosphorylation, subsequently inhibiting E2F transcriptional activity. Consequently, the expression of the E2F target genes (CCNA2, CCNE1, POLA1, and TK-1) was markedly suppressed in nilotinib and AT9283-treated A375P cells. In summary, our findings suggest that BCR-ABL tyrosine kinase inhibitors may regulate the G1-to-S transition in human melanoma A375P cells by modulating the RB-E2F complex.

Frequencies of BCR::ABL1 Transcripts in Patients with Chronic Myeloid Leukemia: A Meta-Analysis.

Chronic myeloid leukemia (CML) is associated with the Philadelphia chromosome and distinct BCR::ABL1 gene transcripts. We assessed the frequencies of these transcripts in Mexico, Latin America, and worldwide. We determined the prevalence of BCR::ABL1 transcripts in CML patients and intercontinental or regional variations using specialized databases and keywords. We analyzed 34 studies from 20 countries, encompassing 5795 patients. Keyword-based searches in specialized databases guided data collection. ANOVA was employed for transcript distribution analysis. The b3a2 transcript was most prevalent globally, followed by b2a2, with e1a2 being the least frequent. Interestingly, Mexico City exhibited a higher incidence of b2a2, while b3a2 predominated in the remaining country. Overall, no significant intercontinental or regional variations were observed. b3a2 was the most common BCR::ABL1 transcript worldwide, with b2a2 following closely; e1a2 was infrequent. Notably, this trend remained consistent in Mexico. Evaluating transcript frequencies holds clinical relevance for CML management. Understanding the frequency of transcript informs personalized CML treatments.

Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

Lipopolymer mediated siRNA delivery targeting aberrant oncogenes for effective therapy of myeloid leukemia in preclinical animal models.

The clinical development of tyrosine kinase inhibitors (TKI) has led to great strides in improving the survival of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. But even the new generation TKIs are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative therapeutic approaches. In contrast to exploiting proteins as targets like most conventional drugs and TKIs, RNA Interference (RNAi) exerts its therapeutic action towards disease-driving aberrant genes. To realize the potential of RNAi, the major challenge is to efficiently deliver the therapeutic mediator of RNAi, small interfering RNA (siRNA) molecules. In this study, we explored the feasibility of using aliphatic lipid (linoleic acid and lauric acid)-grafted polymers (lipopolymers) for the delivery of siRNAs against the FLT3 oncogene in AML and BCR-ABL oncogene in CML. The lipopolymer delivered siRNA potently suppressed the proliferation AML and CML cells via silencing of the targeted oncogenes. In both AML and CML subcutaneous xenografts generated in NCG mice, intravenously administered lipopolymer/siRNA complexes displayed significant inhibitory effect on tumor growth. Combining siFLT3 complexes with gilteritinib allowed for reduction of effective drug dosage, longer duration of remission, and enhanced survival after relapse, compared to gilteritinib monotherapy. Anti-leukemic activity of siBCR-ABL complexes was similar in wild-type and TKI-resistant cells, and therapeutic efficacy was confirmed in vivo through prolonged survival of the NCG hosts systemically implanted with TKI-resistant cells. These results demonstrate the preclinical efficacy of lipopolymer facilitated siRNA delivery, providing a novel therapeutic platform for myeloid leukemias.

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

Zinc as a potential regulator of the BCR-ABL oncogene in chronic myelocytic leukemia cells.

BACKGROUND: Generally, decreased zinc in the serum of tumor patients but increased zinc in tumor cells can be observed. However, the role of zinc homeostasis in myeloid leukemia remains elusive. BCR-ABL is essential for the initiation, maintenance, and progression of chronic myelocytic leukemia (CML). We are currently investigating the association between zinc homeostasis and CML. METHODS: Genes involved in zinc homeostasis were examined using three GEO datasets. Western blotting and qPCR were used to investigate the effects of zinc depletion on BCR-ABL expression. Furthermore, the effect of TPEN on BCR-ABL promoter activity was determined using the dual-luciferase reporter assay. MRNA stability and protein stability of BCR-ABL were assessed using actinomycin D and cycloheximide. RESULTS: Transcriptome data mining revealed that zinc homeostasis-related genes were associated with CML progression and drug resistance. Several zinc homeostasis genes were affected by TPEN. Additionally, we found that zinc depletion by TPEN decreased BCR-ABL mRNA stability and transcriptional activity in K562 CML cells. Zinc supplementation and sodium nitroprusside treatment reversed BCR-ABL downregulation by TPEN, suggesting zinc- and nitric oxide-dependent mechanisms. CONCLUSION: Our in vitro findings may help to understand the role of zinc homeostasis in BCR-ABL regulation and thus highlight the importance of zinc homeostasis in CML.

Prognostic Factors in Philadelphia Chromosome-Positive Acute Myeloid Leukemia Using Fluorescence in Situ Hybridization.

BACKGROUND: Philadelphia chromosome-positive acute myeloid leukemia (Ph+ AML) is a rare leukemia subtype first classified by the World Health Organization in 2016. The incidence of Ph+ AML is approximately 0.5 - 3%, and its prognosis is poor. Ph+ AML with additional chromosomal abnormalities in children has rarely been reported, and its treatment and prognosis remain uncertain. METHODS: We retrospectively analyzed 649 patients with AML from 2006 - 2021. Six (0.9%) patients with Ph+ AML were identified and treated with conventional chemotherapy. The clinical features and prognoses were retrospectively analyzed. RESULTS: Six cases of AML with a Ph chromosome were reported. One of the six individuals exhibited a biphenotypic immunophenotype, one exhibited a simple myeloid immunophenotype, and the other four exhibited myeloid and lymphoid expression. Karyotypic analysis (R banding) was performed in six cases, four of which were classical Ph chromosomal abnormalities, two of which had additional abnormalities outside the Ph chromosome. Fluorescence in situ hybridization (FISH) analysis using the BCR/ABL fusion gene distinguished that the BCR major breakpoint break in three cases was type P210 and the BCR minor breakpoint break in three cases was type P190. The complete remission rate of the six patients in this study using conventional chemotherapy was 60%, with a median survival time of 7.5 months. CONCLUSIONS: In summary, Ph+ AML is a heterogeneous disease often associated with additional chromosomal abnormalities. Ph+ AML is seen with a lymphoid immunophenotype and alterations in associated genes such as the IGH gene. Adults were predominantly P210 and two cases in children were both P190. Conventional treatments are less effective, and there are no standard treatment regimens.

Late relapse of chronic myeloid leukemia after allogeneic bone marrow transplantation points to KANSARL (KANSL1::ARL17A) alteration: a case report with insights on the molecular landscape.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome and the consequent BCR::ABL1 oncoprotein. In the era before the introduction of tyrosine kinase inhibitors (TKIs), the only potentially curative treatment was allogeneic hematopoietic stem cell transplantation (HSCT). Here, we present the case of a patient affected by CML who experienced a relapse 20 years after allogeneic HSCT. Following relapse, the patient was treated with imatinib and bosutinib, resulting in a deep molecular response and successfully discontinued treatment. Additional analysis including whole-exome sequencing and RNA sequencing provided some insights on the molecular mechanisms of the relapse: the identification of the fusion transcript KANSL1::ARL17A (KANSARL), a cancer predisposition fusion gene, could justify a condition of genomic instability which may be associated with the onset and/or probably the late relapse of his CML.

The Mozart effect in chronic myeloid leukaemia.

Clinical research has not been able to establish whether the differences between first- and second-generation BCR-ABL 1 kinase inhibitors are clinically relevant with regard to outcome. In the study by Alcazer et al., a relevant difference seems to emerge-paradoxically in the absence of the drugs-as demonstrated by differences in the relapse kinetics after cessation of therapy. Commentary on: Alcazer et al. Kinetics of molecular recurrence after tyrosine kinase inhibitor cessation in chronic phase chronic myelogenous leukaemia patients. Br J Haematol 2024;204:1536-1539.

Navigating the Management of Chronic Phase CML in the Era of Generic BCR::ABL1 Tyrosine Kinase Inhibitors.

Over the past several years, advances in research, treatment, and market dynamics have impacted treatment strategies in chronic myeloid leukemia in chronic phase (CML-CP). They include the broader availability of cost-effective generic imatinib, and soon other generic second-generation tyrosine kinase inhibitors (TKIs). Access to affordable generics means that all patients with CML-CP should have access to safe and highly effective lifelong therapies. When overall survival is the treatment endpoint, imatinib provides a good treatment value. Second-generation TKIs may be the best frontline strategy when treatment-free remission is the goal. Recent studies have shown maintained efficacy and reduced toxicity when TKIs are used at reduced dosing. Reduced-dose schedules of second-generation TKIs (which are less toxic and induce faster deep molecular responses) may render generic second-generation TKIs a more attractive treatment option. Adjusting the dose of TKI in the presence of mild-to-moderate, or even severe but reversible, adverse events may be preferable to switching to a different TKI. The selection of second-line and beyond therapies depends on the evolving patterns observed with frontline treatment. Dose-adjusted ponatinib schedules have demonstrated improved efficacy and safety in patients resistant to second-generation TKIs or those with T315I-mutated disease. For asciminib, longer-term follow-up is needed to better evaluate its safety and efficacy compared with ponatinib. Allogeneic stem cell transplantation represents a valid alternative to newer-generation TKIs, with a better treatment value when TKIs are priced at >$40,000/year.