Development Of Chronic Myeloid Leukemia Research Articles

BCAT1 contributes to the development of TKI-resistant CML.

Although most of chronic myeloid leukemia (CML) patients can be effectively treated by the tyrosine kinase inhibitors (TKIs), such as Imatinib, TKI-resistance still occurs in approximately 15-17% of cases. Although many studies indicate that branched chain amino acid (BCAA) metabolism may contribute to the TKI resistance in CML, the detailed mechanisms remains largely unknown. The cell proliferation, colony formation and in vivo transplantation were used to determined the functions of BCAT1 in leukemogenesis. Quantitative real-time PCR (RT-PCR), western blotting, RNA sequencing, BCAA stimulation in vitro were applied to characterize the underlying molecular mechanism that control the leukemogenic activity of BCAT1-knockdown cells. In this report, we revealed that branched chain amino acid transaminase 1 (BCAT1) is highly enriched in both mouse and human TKI-resistant CML cells. Leukemia was almost completely abrogated upon BCAT1 knockdown during transplantation in a BCR-ABLT315I-induced murine TKI-resistant CML model. Moreover, knockdown of BCAT1 led to a dramatic decrease in the proliferation of TKI-resistant human leukemia cell lines. BCAA/BCAT1 signaling enhanced the phosphorylation of CREB, which is required for maintenance of TKI-resistant CML cells. Importantly, blockade of BCAA/BCAT1 signaling efficiently inhibited leukemogenesis both in vivo and in vitro. These findings demonstrate the role of BCAA/BCAT1 signaling in cancer development and suggest that targeting BCAA/BCAT1 signaling is a potential strategy for interfering with TKI-resistant CML.

Mutations in Genes Producing Nitric Oxide and Hydrogen Sulfide and Their Connection With Apoptotic Genes in Chronic Myeloid Leukemia.

Background Despite advances in chronic myeloid leukemia (CML) genetics, the role of nitric oxide (NO) and hydrogen sulfide (H2S) gene mutations and their relationship to apoptotic genes is unclear. Therefore, this study investigated NO- and H2S-producing genes' mutations and their interactions with apoptotic genes using Sanger sequencing and next-generation sequencing (NGS). Methodology A complete blood count (CBC) was carried out to measure the total number of white blood cells, while IL-6 levels were assessed in both control and CML patients using an ELISA technique.Sanger sequencing was used to analyzemutations in the CTH and NOS3 genes, whereas NGS was applied to examine mutations on all chromosomes. Results White blood cell (WBC) and granulocyte counts were significantly higher in CML patients compared to controls (p<0.0001), and monocyte counts were similarly higher (p<0.05). Interleukin-6 (IL-6) levels were significantly elevated in CML patients thancontrols (p<0.0001), indicating a possible link to CML etiology or progression. Multiple mutations have been identified in both genes, notably in CTH exon 12 and the NOS3genes VNTR, T786C, and G894T. This study also measured IL-6 concentrations using IL-6 assays, identifying its potential as a CML prognostic diagnostic. WBC counts, granulocyte counts, and mid-range absolute counts, or MID counts, were significantly higher in CML patients than in normal control individuals. NGS identified 1643 somatic and sex chromosomal abnormalities and 439 actively expressed genes in CML patients. The findings imply a genomic landscape beyond the BCR-ABL1 mutation in CML development compared to other databases. Conclusion In conclusion, this study advances the understanding of the genetic characteristics of CML by identifying mutations in the NO- and H2S-producing genes and their complex connections with genes involved in apoptosis. The comprehensive genetic profile obtained by Sanger sequencing and NGS provides possibilities for identifying novel targets for therapy and personalized treatments for CML, therefore contributing to developments in hematological diseases.

CRKL but not CRKII contributes to hemin-induced erythroid differentiation of CML.

Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.

STUDYING THE AGE OF ONSET AND DETECTION OF CHRONIC MYELOID LEUKEMIA USING A THREE-STAGE STOCHASTIC MODEL

Chronic Myeloid Leukemia (CML) is a biphasic malignant clonal disorder that progresses, first with a chronic phase, where the cells have enhanced proliferation only, and then to a blast phase, where the cells have the ability of self-renewal. It is well recognized that the Philadelphia chromosome (which contains the BCR-ABL fusion gene) is the “hallmark of CML”. However, empirical studies have shown that the mere presence of BCR-ABL may not be a sufficient condition for the development of CML, and further modifications related to tumor suppressors may be necessary. Accordingly, we develop a three-mutation stochastic model of CML progression, with the three stages corresponding to the non-malignant cells with BCR-ABL presence, the malignant cells in the chronic phase, and the malignant cells in the blast phase. We demonstrate that the model predictions agree with age incidence data from the United States. Finally, we develop a framework for the retrospective estimation of the time of onset of malignancy, from the time of detection of the cancer.

Abstract 2307: State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML)

Abstract Chronic myeloid leukemia (CML) is initiated and initially maintained by BCR-ABL which is clinically targeted using tyrosine kinase inhibitors (TKIs). Although TKIs can induce long-term remission, they are frequently not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. We collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR-ABL-inducible transgenic mice that recapitulates human chronic phase CML and wild-type controls. From the transcriptome, we constructed a CML state-space and a three-well leukemogenic potential landscape that describes CML state-transition from health to disease. The potential’s stable critical points were used to define three distinct disease states. The Early state was characterized by anti-CML genes (n=35) that opposed leukemia progression, whereas an expanding number of pro-CML genes characterized the Transition state (n=357) and Late states (n=1,858). Gene modules that were co-regulated at the unstable points in the potential landscape were identified as the drivers of transition between stable disease states. To investigate CML therapies we used two treatments: silencing of BCR/ABL by readministering Tet (Tet-on, Tet-off [TOTO]) which represented a best-case scenario of disease being cured, and TKI (nilotinib) therapy which represents current clinical practice. TOTO returned the diseased mice transcriptomes to a near health state, without reaching it, suggesting partly irreversible transformation. TKI, however, only reverted the transcriptome to an intermediate disease state, without approaching health, and disease relapse occurred soon after treatment. Finally, using only the earliest time-point as initial conditions, our state-transition models accurately predicted both disease progression and treatment response to both therapies for each individual mouse. In conclusion, these results show state-transition analysis is a valuable approach to gain real time insights into CML development, progression and ongoing treatment response that can also be applied to other type of leukemia and cancers in general. We predicted disease evolution and treatment response in a murine model that recapitulates human disease, supporting this as a potentially valuable approach to time clinical intervention even before phenotypic changes become detectable. Citation Format: David E. Frankhouser, Russell C. Rockne, Dandan Zhao, Sergio Branciamore, Lisa Uechi, Denis O'Meally, Yu-Hsuan Fu, Ya-Huei Kuo, Bin Zhang, Guido Marcucci. State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2307.

Risk Factors and Prognosis of Chronic Myeloid Leukemia in Saudi Arabia: A Systematic Review

Background: Among adult leukaemias, chronic myeloid leukaemia (CML) is one of the most prevalent types. The Philadelphia chromosome, which constitutively activates tyrosine kinase through the BCR-ABL1 oncoprotein, is central to the pathophysiology of CML.&#x0D; Objectives: To study the risk factors and prognosis of chronic myeloid leukemia in Saudi Arabia.&#x0D; Methods: PubMed, SCOPUS, Web of Science, and Science Direct were systematically searched for relevant literature. Rayyan QRCI was employed throughout this comprehensive process.&#x0D; Results and Interpretation: seven studies with a total of 658 patients and 357 (54.3) were males. Risk factors for the development of CML were shown to be limited to younger and middle-aged groups, with a slight male predominance. This study showed that imatinib for CML has both long-term efficacy and manageable side effects. Improved compliance, lower blast and basophil counts, increased haemoglobin, increased platelets, and a low-risk score were all associated with different molecular reactions to imatinib. Future research is required to assess the possible risk factors, management lines, and prognostic factors of CML in Saudi population.

Thrombosis and Bleeding Outcomes Among Veterans with Chronic Myeloid Leukemia and Association with Agent Orange Exposure

The incidence of chronic myeloid leukemia (CML) in the United States has been reported to be 1.75/100,000. Older age and male patients are more likely to be diagnosed with CML (Chen Y et al. Trends in chronic myeloid leukemia incidence and survival in the United States from 1975 to 2009. Leuk Lymphoma. 2013 Jul;54(7):1411-7). Since their introduction in 2000, the mainstay of treatment for CML has been tyrosine kinase inhibitors (TKIs). These medications are generally well-tolerated. Of the TKIs in use today, Ponatinib is known to have adverse vascular events but is not recommended for use in the first line. Nilotinib has also been shown to have adverse arterial events, which tend to occur late in the treatment course (Rea D. Management of adverse events associated with tyrosine kinase inhibitors in chronic myeloid leukemia. Ann Hematol. 2015 Apr;94 Suppl 2:S149-58.). In this study, we evaluated the thrombosis and bleeding complications of CML in the era of TKIs. The Sergeant First Class (SFC) Heath Robinson Honoring our Promise to Address Comprehensive Toxics (PACT) Act is a law that expanded Veterans' health care and benefits to those with exposure to carcinogens such as Agent Orange (AO). The PACT Act has a list of presumptive conditions due to exposure to AO. Myeloid malignancies have not been added to the presumptive list due to insufficient evidence from the Veterans and Agent Orange: Update 11 (2018). We also investigated the association of CML and AO exposure. We performed a case control study from January 1, 2006 - January 26, 2023, by utilizing the Veterans Affairs Informatics and Computing Infrastructure (VINCI) database to characterize the association of AO exposure with development of CML. ICD-9 and -10 codes identified Veterans with CML, arterial thrombosis (AT), venous thrombosis (VT), and bleeding. Age-, sex-, and race-matched controls (1:1) were selected from VINCI, who are Veterans without CML from Illinois, the state most representative of the U.S. population. Exposure to Agent Orange was self-reported and verified through Veterans' duration and location of service. We stratified the Veterans according to birth year (born before or after 1960) as those born before 1960 are most likely to have AO exposure. Qualitative data were compared by chi-square tests. When comparing all Veterans with CML and Veterans without CML, AO exposure is associated with the development of CML, OR 1.71, 95% CI 1.63-1.80, p&amp;lt;0.0001 (Figure 1). When adjusting for birth year and comparing all Veterans with CML unmatched controls from Illinois, there remains a statistically significant association, OR 1.21, 95% CI 1.14-1.28, p&amp;lt;0.0001. Further comparison to a group of matched controls is also statistically significant, OR 1.14, 95% CI 1.06-1.24, p=0.0007. Veterans born before 1960 and Males were significantly associated with development of CML, OR 6.25 95% CI 5.89-6.63 and OR 4.79 95% CI 4.29-5.36, both p&amp;lt;0.0001. Table 1 details the demographics and clinical characteristics among Veterans with or without CML, with or without AO exposure, and matched and unmatched controls. When comparing all Veterans with CML and matched controls, we observed an association of CML with bleeding (OR 3.95, 95% CI 3.70-4.22), AT (OR 2.58, 95% CI 2.43-2.74), and VT (OR 2.19, 95% CI 1.98-2.41), all p&amp;lt;0.0001. We then investigated whether AO exposure increases the risk of AT, VT, and among Veterans with CML. When adjusting for birth year, we observed an increased risk of AT (OR 1.35, 95% CI 1.21-1.51, p&amp;lt;0.0001) and bleeding (OR 1.13, 95% CI 1.02-1.26, p=0.02) among Veterans with CML with AO exposure but not VT (OR 1.17, 95% CI 0.99-1.37, p=0.05). Our analysis demonstrates a positive association between previous exposure to AO and subsequent development of CML in Veterans. These findings suggest that CML is the second myeloid malignancy to be associated with AO exposure apart from myeloproliferative neoplasms (J Clin Oncology 2023 41:16_suppl, 7011). We also demonstrate a strong association in patients with CML and outcomes including AT, VT, and bleeding. This is the first large cohort study to demonstrate these associations. These outcomes may be affected by tyrosine kinase inhibitors for the treatment of CML. Further investigation will include overall survival data, specific prognostic factors related to CML, cardiovascular risk factors, and information on the timing of AT, VT, and bleeding in relation to disease diagnosis and treatment onset.

Uncovering the Transcriptional Landscape of CML Development and Therapeutic Interventions Via State-Transition Modeling

Chronic Myeloid Leukemia (CML) is driven by expression of the BCR-ABL fusion gene. Despite the success of treatment with tyrosine kinase inhibitors (TKIs), the disease-originating leukemic stem cells frequently persist through therapy, and disease cannot be eradicated. Thus, new biomarkers beyond BCR/ABL may be necessary to assess disease state and treatment response, and to identify novel targets and therapeutics that could lead to a cure. Herein, we used time-series RNA-seq to molecularly characterize the transcriptional states of CML and predict disease evolution and treatment response. Using weekly blood draws from a murine tetracycline (Tet)-inducible BCR/ABL expression model of chronic phase CML and non-BCR/ABL expressing controls ( Fig 1A), we applied state-transition theory to mathematically model CML development. We constructed a CML state-space using dimensionality reduction to the full transcriptomic data of the time-series CML and control mice. From the sample distribution in the state-space, we empirically derived healthy (no BCR/ABL) and perturbed (BCR/ABL-induced) potentials ( Fig 1B). Using these potentials, our model described the transcriptome evolution during CML development as a particle undergoing Brownian motion. We used the Fokker-Planck (FP) solution of the Langevin equation of motion to both accurately model the transcriptome and predict the future dynamics from a single sample used as an initial condition. While control mice remain in the region of the state-space corresponding to health, each of the BCR/ABL expressing mice moved toward CML state as early as one week after BCR/ABL induction and before detection of BCR/ABL mRNA expression. Of note, no significant difference was observed between the experimental and the FP predicted trajectories and survivals, supporting the accuracy of our approach in predicting disease evolution and outcome at the earliest time points. The CML potential was described as a three well potential with three stable (Early c1, Transition c3, Late c5) and two unstable (Early Transition c2 and Late Transition c4). Differentially expressed genes (DEGs) corresponding to Early c1, Transition c3, Late c5 states were identified by comparing with control mice. Importantly, the state-space model also allowed assessment of whether each DEG had a pro- or anti-CML effect. At the Early state c1, we identified 78 DEGs; 55% of them were anti-CML, indicating an early attempt of the biological system to prevent disease development. By GSEA, we showed that the anti-CML Hallmark gene sets included EMT, angiogenesis, hypoxia and the only pro-CML gene set was IL2/Stat5. In contrast, at the Transition c3 and Late c5 states, we identify 366 and 1858 DEGs, respectively; nearly all these DEGs had a pro-CML effect and were enriched for Hallmark gene sets involving inflammation, angiogenesis and metabolism. Using CML state-space as a pseudotime ordering of the CML samples, we also studied modules of non-linear gene expression dynamics. This approach revealed changes in gene expression rates (inflection points) coinciding with the unstable critical points (c2 and c4) that separated the otherwise stable states. The genes in these transition modules included immune/inflammation processes and likely represented those genes that drove CML state-transition. To investigate the evolution of disease “in reverse” (i.e., after the CML phenotype developed), we treated two cohorts of CML mice, one with a TKI (nilotinib) for 4 weeks and one with Tet to turn off the expression of BCR/ABL (Tet-off, Tet-on [TOTO]). We adapted the state-transition model to a “treatment state-transition” model by introducing a specific parameter, g, which both accurately described and predicted individual treatment efficacy. We showed that although the circulating leukemic cells were suppressed by TKI, the disease state recovered only from c5 to c3, but never reached the health (control) state, and the treated mice rapidly relapsed once treatment was completed. Conversely, the TOTO cohort, which had the BCR/ABL expression completed turn off, returned stably to the healthy state. In conclusion, our state-transition model provides a theory-based analytic framework for investigating disease dynamics and predicting treatment response and outcome. Importantly, can be translated into the clinic for novel transcriptomic-based diagnostic approaches.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4+/+ and Lama4-/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4-/- recipients showed significantly increased platelet counts in blood compared to Lama4+/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4-/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1+ cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4-/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4-/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4-/- recipients (33.6%) than that in Lama4+/+ recipients (41.5%), raising a possibility that Lama4-/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4-/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Cytogenetic Analysis of Chronic Myeloid Leukemia in an Eastern Indian Population

Chronic myeloid leukaemia (CML) is a common translocation known as BCR-ABL1, also referred to as the Philadelphia chromosome, that affects the primitive hematopoietic stem cell. The GLOBOCAN 2020 report estimates that there will be roughly 19.3 million new cancer diagnoses and 10 million cancer-related deaths in 2020. A fusion oncoprotein called BCR-ABL1, a constitutively active tyrosine kinase that is essential for the development of CML, can be inhibited to slow the course of the illness. The bone marrow and peripheral blood pile up different forms of immature and mature granulocytes or blast cells. In the previous study of CML in India, the annual incidence was reported to be 0.8 to 2.2 per 100,000 population. In leukemia, the most, common type is CML in India. Therefore, chromosomal analysis of CML plays an essential aspect in diagnosing leukemia patients. In the current study, 130 CML cases from the Eastern Indian population ranging in age from 7 to 79 years were cytogenetically analysed (mean of 36). In the study population, the male to female ratio was 1.24:1, with 72 males (55.38%) and 58 women (44.61%) participating. 122 (93.7%) of the 130 cases could be successfully karyotyped, whereas 8 (6.3%) failed culture tests. Out of the 130 cases that were noted, 25 (19.25%) had karyotypes that were normal, while 97 (74.61%) had the Philadelphia (Ph’) chromosome, which has the distinctive translocation t(9;22); Ph’+ve. Furthermore, we conclude that while more advanced molecular techniques cannot entirely replace cytogenetics, which continues to be the focus of laboratory studies into the condition, they can be used in conjunction with it to help diagnose of chronic myeloid leukemia.

Association of ABCB1, ABCG2 drug transporter polymorphisms and smoking with disease risk and cytogenetic response to imatinib in chronic myeloid leukemia patients

BackgroundDespite acceptable results of imatinib in the treatment of chronic myeloid leukemia (CML), some patients fail to acquire a complete cytogenetic response (CCyR), which may be caused by polymorphisms in the pharmacogenetic genes. The study aimed to evaluate the association of two polymorphisms in the ABCB1 and ABCG2 genes with cytogenetic response to imatinib and the risk of CML development. MethodsWe genotyped ABCB1 (c .2677G/T/A) and ABCG2 (c .421C/A) polymorphisms by PCR-RFLP, T-ARMS-PCR methods in 111 patients with CML and 102 sex- and age-matched healthy subjects. CCyR was determined by standard chromosome banding analysis (CBA). ResultsAnalysis of polymorphisms showed significant association of ABCG2 c.421CA genotype (p < 0.0001; OR = 0. 17), and ABCG2c.421A allele (p < 0.0001; OR = 0.31) with decreased risk of CML. Moreover, ABCB1c.2677GT- ABCG2c.421CC combined genotype (p = 0.017; OR = 4.20) was associated with increased risk of CML. Analysis of the joint effect of SNP-smoking combination showed that smoker subjects with the ABCB1c.2677GG/GT (p = 0.001; OR = 15.96, p = 0.001; OR = 8.13, respectively) or ABCG2c.421CC genotypes (p = 0.001; OR = 5.82) had the increased risk of CML, while the risk of the CML in non-smokers carrying the ABCG2c.421CA (p < 0.0001; OR = 0. 18) genotype was strongly decreased compared with reference group. Regarding drug response, ABCG2c.421 CC/CA genotypes in the smoker patients were associated with an increased risk of resistance to imatinib (p < 0.0001; OR = 7.02, p = 0.018; OR = 4.67, respectively). ConclusionOur results suggest the impact of ABCG2c .421C/A polymorphism on CML development, and smoking may have a synergistic role in the risk of CML and resistance to imatinib.

MTORC2 Is Activated under Hypoxia and Could Support Chronic Myeloid Leukemia Stem Cells.

Hypoxia is a critical condition that governs survival, self-renewal, quiescence, metabolic shift and refractoriness to leukemic stem cell (LSC) therapy. The present study aims to investigate the hypoxia-driven regulation of the mammalian Target of the Rapamycin-2 (mTORC2) complex to unravel it as a novel potential target in chronic myeloid leukemia (CML) therapeutic strategies. After inducing hypoxia in a CML cell line model, we investigated the activities of mTORC1 and mTORC2. Surprisingly, we detected a significant activation of mTORC2 at the expense of mTORC1, accompanied by the nuclear localization of the main substrate phospho-Akt (Ser473). Moreover, the Gene Ontology analysis of CML patients' CD34+ cells showed enrichment in the mTORC2 signature, further strengthening our data. The deregulation of mTOR complexes highlights how hypoxia could be crucial in CML development. In conclusion, we propose a mechanism by which CML cells residing under a low-oxygen tension, i.e., in the leukemia quiescent LSCs, singularly regulate the mTORC2 and its downstream effectors.

Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML.

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 109 and 2.61 × 109IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.

Medium-mass Molecules in Plasma of Patients at Different Stages of Chronic Myeloid Leukemia Progression

Chronic myeloid leukemia (CML) is a clonal tumor disease caused by malignant transformation of hematopoietic stem cells, in which a chimeric BCR-ABL gene is formed as a result of specific translocation of areas between the 9th and 22nd chromosomes. This genetic «defect» ensures the uncontrolled proliferation of myeloid cells. The objective: to analyse the content of medium-mass molecules (MMM) in peripheral venous blood plasma of patients with CML at different stages of the disease and theoretical justification of approaches to the correction of detected changes. Materials and methods. The study enrolled 77 patients with CML, who were divided into three groups according to the stage of disease. The control group included 20 people who were primary donors of the Kyiv City Blood Center. The main parameters characterizing the state of protein and carbohydrate metabolism were analyzed. All patients groups were similar in terms of age, sex, anthropometric parameters, and duration of the disease. All patients during hospitalization were examined by clinical, laboratory, instrumental and special research methods, and if necessary, consulted by specialists. Statistical processing of the results was done by methods of variational statistics, using the Microsoft Excel XP computer program. Results. Today, there are known factors that determine the development of CML, namely: chromosomal abnormalities, ionizing radiation, hematopoietic system diseases, toxic effects of drugs and polluted environment, etc. Changes in the content of MMM in plasma of patients with CML compared to the control group were determined. As the disease progresses, the imbalance of MMM metabolism deepens. The severity of plasma MMM content violations of CML patients is accompanied by endogenous body intoxication, since MMM are universal markers of endogenous metabolic intoxication. Conclusions. Changes in the content of medium-mass molecules in the plasma of patients with chronic myeloid leukemia dictate the need for further in-depth research to determine role in the pathophysiology of the disease for further treatment and reduction of the manifestations of endogenous intoxication. Effective treatment of patients with CML is based on modern diagnostics, timely prescription of drugs and subsequent monitoring of the course of the disease.

Histone chaperone ASF1A accelerates chronic myeloid leukemia blast crisis by activating Notch signaling

The blast crisis (BC) is the final deadly phase of chronic myeloid leukemia (CML), which remains a major challenge in clinical management. However, the underlying molecular mechanism driving blastic transformation remains unclear. Here, we show that ASF1A, an essential activator, enhanced the transformation to CML-BC by mediating cell differentiation arrest. ASF1A expression was aberrantly increased in bone marrow samples from CML-BC patients compared with newly diagnosed CML-chronic phase (CP) patients. ASF1A inhibited cell differentiation and promoted CML development in vivo. Mechanistically, we identified ASF1A as a coactivator of the Notch transcriptional complex that induces H3K56ac modification in the promoter regions of Notch target genes, and subsequently enhanced RBPJ binding to these promoter regions, thereby enhancing Notch signaling activation to mediate differentiation arrest in CML cells. Thus, our work suggests that targeting ASF1A might represent a promising therapeutic approach and a biomarker to detect disease progression in CML patients.

Cell Contact with Endothelial Cells Favors the In Vitro Maintenance of Human Chronic Myeloid Leukemia Stem and Progenitor Cells

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells.

DNA- and RNA-Binding Proteins Linked Transcriptional Control and Alternative Splicing Together in a Two-Layer Regulatory Network System of Chronic Myeloid Leukemia.

DNA- and RNA-binding proteins (DRBPs) typically possess multiple functions to bind both DNA and RNA and regulate gene expression from more than one level. They are controllers for post-transcriptional processes, such as splicing, polyadenylation, transportation, translation, and degradation of RNA transcripts in eukaryotic organisms, as well as regulators on the transcriptional level. Although DRBPs are reported to play critical roles in various developmental processes and diseases, it is still unclear how they work with DNAs and RNAs simultaneously and regulate genes at the transcriptional and post-transcriptional levels. To investigate the functional mechanism of DRBPs, we collected data from a variety of databases and literature and identified 118 DRBPs, which function as both transcription factors (TFs) and splicing factors (SFs), thus called DRBP-SF. Extensive investigations were conducted on four DRBP-SFs that were highly expressed in chronic myeloid leukemia (CML), heterogeneous nuclear ribonucleoprotein K (HNRNPK), heterogeneous nuclear ribonucleoprotein L (HNRNPL), non-POU domain–containing octamer–binding protein (NONO), and TAR DNA-binding protein 43 (TARDBP). By integrating and analyzing ChIP-seq, CLIP-seq, RNA-seq, and shRNA-seq data in K562 using binding and expression target analysis and Statistical Utility for RBP Functions, we discovered a two-layer regulatory network system centered on these four DRBP-SFs and proposed three possible regulatory models where DRBP-SFs can connect transcriptional and alternative splicing regulatory networks cooperatively in CML. The exploration of the identified DRBP-SFs provides new ideas for studying DRBP and regulatory networks, holding promise for further mechanistic discoveries of the two-layer gene regulatory system that may play critical roles in the occurrence and development of CML.

The genetics of chronic myeloid leukaemia and recent advancements in therapy

Each year, approximately 750 people (commonly >65 years) are diagnosed with leukaemia in the United Kingdom (UK). Of every 100 cases of leukaemia, 15 are diagnosed with Chronic Myeloid Leukaemia (CML), also referred to as chronic myelogenous leukaemia. CML is a form of cancer affecting immature white blood cells (i.e. leukocytes). Individuals with CML present with a raised white blood cell count. CML is characterised by the unregulated growth of myeloid cells arising from a transformed haematopoietic stem cell (HSC). Leukocytes serve vital roles in the innate immune system. For example, neutrophils (a type of granulocyte) are highly mobile and first responders in inflammation. Neutrophils are activated for phagocytosis or the release of cytokines, and recent studies have shown their role in neutrophil extracellular traps. This article outlines the pathophysiological mechanisms and genetic basis underlying the development of CML. It also highlights recent advancements in targeted therapies to combat disease severity and progression.

Allogeneic stem cell transplantation for chronic myeloid leukemia in the tyrosine kinase inhibitor era

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm characterized by the production of mature granulocytes and their precursors that are dysregulated and uncontrolled. The presence of the Philadelphia chromosome (Ph) is the hallmark of CML; it encodes a chimeric protein with constitutive tyrosine kinase activity that leads to uncontrolled cell growth and eventually the development of CML. Tyrosine kinase inhibitors (TKI) revolutionized the management of patients with CML. Before TKIs, hematopoietic stem cell transplantation had a major role in the treatment of these patients, but currently, its use is limited to cases presenting in the advanced phase and patients in the chronic phase failing multiple TKIs. In this article, the author summarizes the data about hematopoietic stem cell transplantation use in chronic phase CML, reviews the published guidelines, and provides his opinion.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia

AbstractLipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL–AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.