Chronic Myeloid Leukemia Stem Cells Research Articles

Predictive nonlinear modeling of malignant myelopoiesis and tyrosine kinase inhibitor therapy.

Chronic myeloid leukemia (CML) is a blood cancer characterized by dysregulated production of maturing myeloid cells driven by the product of the Philadelphia chromosome, the BCR-ABL1 tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have proved effective in treating CML, but there is still a cohort of patients who do not respond to TKI therapy even in the absence of mutations in the BCR-ABL1 kinase domain that mediate drug resistance. To discover novel strategies to improve TKI therapy in CML, we developed a nonlinear mathematical model of CML hematopoiesis that incorporates feedback control and lineage branching. Cell-cell interactions were constrained using an automated model selection method together with previous observations and new in vivo data from a chimeric BCR-ABL1 transgenic mouse model of CML. The resulting quantitative model captures the dynamics of normal and CML cells at various stages of the disease and exhibits variable responses to TKI treatment, consistent with those of CML patients. The model predicts that an increase in the proportion of CML stem cells in the bone marrow would decrease the tendency of the disease to respond to TKI therapy, in concordance with clinical data and confirmed experimentally in mice. The model further suggests that, under our assumed similarities between normal and leukemic cells, a key predictor of refractory response to TKI treatment is an increased maximum probability of self-renewal of normal hematopoietic stem cells. We use these insights to develop a clinical prognostic criterion to predict the efficacy of TKI treatment and design strategies to improve treatment response. The model predicts that stimulating the differentiation of leukemic stem cells while applying TKI therapy can significantly improve treatment outcomes.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

Abstract A013: Sex-dependent hematopoietic stem cell aging and leukemogenic potential

Abstract Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders. But how HSC aging and sex co-influence leukemogenesis is poorly understood. By examining HSC aging in young, aging (75% lifespan) and longevity (110% lifespan) mice in males vs females, we have discovered that aging males have a significantly higher HSC frequency than females, and that male HSC aging is characterized by an expansion of Lin-CD150+SP HSCs and a decrease of Lin-CD150-SP HSCs, whereas females show opposite HSC changes towards aging and longevity. In line with HSC changes, aging males exhibit an increase of white blood cell counts that is primarily driven by increased myeloid cells, whereas aging females shows a decrease of white blood cell counts that is primarily driven by reduced lymphoid cells. To understand leukemogenic potential of aging HSCs in the true setting of organismal aging, we generated the first aging mouse model of chronic myeloid leukemia (CML) induced by BCR-ABL1 (Oncogene, 2021, 40: 3152-3163). We found that aging male HSCs are significantly more susceptible to BCR-ABL1 transformation and CML induction in aging male mice than aging female HSCs for CML induction in aging female mice. However, in the absence of oncogenic translocation genes, longevity female but not male mice rapidly develop B/myeloid mixed phenotype acute leukemia, a disease of HSC origin, in primary bone marrow transplantation recipients. Leukemias in our aging mouse models phenocopy those of humans in that the incidence of myeloid leukemia is higher in aging men than in women overall, but longevity women have a higher incidence of myeloid leukemia. Using single cell RNA sequencing analysis of HSCs, we found distinct cell clustering patterns and signaling pathways of aging HSCs between male and female mice. The top pathways seen in male HSC aging involve TGFβ and TNFα signaling that have been linked to poor drug response and persistence of human CML stem cells, while those for female HSC aging involve IFNα/γ signaling pathways that have been associated with good drug response of human CML stem cells. Together, our results demonstrate sex-dependent HSC aging and leukemogenic potential and that molecular changes in aging HSCs can inform drug response of leukemia stem cells. Our findings provide novel insight into the etiology of leukemia in males vs females and suggest potential benefit to use sex-based prevention and therapeutic strategies for leukemia in men vs women. Citation Format: WenYong Chen. Sex-dependent hematopoietic stem cell aging and leukemogenic potential [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A013.

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.

Novel therapeutic strategy for targeting chronic myeloid leukemia stem cells via IRAK1/4 inhibition

Chronic myeloid leukemia (CML) stem cells have been identified to promote CML relapse due to their quiescent cell cycle and tyrosine kinase inhibitor resistance. Therefore, their eradication is important for the cure of CML. We herein identified the quiescent CML stem cell fraction using a G0 marker that can visualize quiescent cells. Whole-transcriptome analysis of imatinib-resistant, quiescent CML stem cells revealed that NF-κB is activated via inflammatory signals in the same cells. The combination of imatinib and an inhibitor of this inflammatory signal (IRAK1/4 inhibitor) effectively eliminated CML stem cells and attenuated PD-L1 expression in CML stem cells. Furthermore, the combination of anti-PD-L1 antibody and imatinib effectively eliminated CML stem cells in the presence of T-cell immunity, indicating the importance of creating an environment in which T cells can attack CML stem cells. Thus, IRAK1/4 inhibitors exert two effects: blocking CML stem cell survival and proliferation signals by inhibiting NF-κB and blocking T cell immune evasion by reducing PD-L1 expression in CML stem cells. Collectively, their combination may be one of the attractive strategies for achieving a radical cure for CML. Discussions regarding the possibility of future medications seem warranted.

Targeting BCR-ABL1-positive leukaemias: a review article.

Treatment and understanding of BCR::ABL1-positive leukaemias is a precision medicine success story. Our appreciation of the BCR::ABL1 gene and resulting BCR::ABL1 oncoprotein in chronic myeloid leukaemia (CML) and Philadelphia chromosome-positive (Ph+) acute leukaemias, has led to treatment advances associated with exceptional improvements in patient outcomes with normal life expectancy for many patients with chronic phase (CP-)CML. However, despite these major therapeutic advances, the management of Ph+ leukaemias remains complex, with development of specific resistance mutations on treatment, as well as the need for lifelong therapy in most patients due to the persistence of CML stem cells despite prolonged tyrosine kinase inhibitors (TKIs) treatment. BCR::ABL1-specific TKIs are associated with chronic toxicities affecting quality-of-life in many patients but can also result in more serious pulmonary and cardiovascular complications. Dose optimisation is increasingly being used to manage side effects and maintain molecular response in CML patients. Here, we review the development of BCR::ABL1-specific TKIs from the discovery of imatinib in 1996 to the more recent second- and third-generation TKIs and emerging specifically targeting the ABL myristoyl pocket (STAMP) inhibitors. We will also evaluate the current evidence for treatment of BCR::ABL1-positive leukaemias, including TKI discontinuation in optimally responding CP-CML patients.

YBX1 regulates the survival of chronic myeloid leukemia stem cells by modulating m6A-mediated YWHAZ stability.

Chronic myeloid leukemia (CML) is a myeloproliferative disease derived from hematopoietic stem cells (HSCs) that harbor Philadelphia chromosome (Ph chromosome). In clinic, leukemia stem cells (LSCs) in CML are insensitive to the treatment with tyrosine kinase inhibitors, and are responsible for disease relapse. However, the molecular mechanisms for maintaining LSCs survival remain elusive. CML patient-derived cell lines and BCR-ABL-induced CML mouse model were used to explore the role of YBX1 in regulating the survival of CML LSCs. Bone marrow transduction and transplantation, and colony-forming unit assay were used to investigate LSC function. The underlying mechanism of how YBX1 regulates LSCs survival were assessed using flow cytometry, RNA sequencing, western blot, RNA decay assay, co-immunoprecipitation and RNA immunoprecipitation. Here we show that RNA-binding protein YBX1 plays an important role in regulating survival of CML LSCs. We find that YBX1 expression is significantly increased in CML cells, and confirm that YBX1 is required for maintaining survival of LSCs. Deletion of YBX1 impairs the propagation of CML through blocking cell proliferation and inducing apoptosis of LSCs. Mechanistically, we find that YBX1 regulates expression of apoptotic associated genes. YBX1 cooperates with RNA m6A reader IGF2BPs to stabilize YWHAZ transcript in an m6A-dependent manner, and loss of YBX1 decreases YWHAZ expression by accelerating mRNA decay. Restoration of YWHAZ efficiently rescues the defects of YBX1-deficient CML cells. Our findings reveal a critical role of YBX1 in maintaining survival of CML LSCs, which provides a rationale for targeting YBX1 in CML treatment.

Integrating Single-Cell Transcriptome and Network Analysis to Characterize the Therapeutic Response of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by a unique BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) were developed to target the BCR-ABL oncoprotein, inhibiting its abnormal kinase activity. TKI treatments have significantly improved CML patient outcomes. However, the patients can develop drug resistance and relapse after therapy discontinues largely due to intratumor heterogeneity. It is critical to understand the differences in therapeutic responses among subpopulations of cells. Single-cell RNA sequencing measures the transcriptome of individual cells, allowing us to differentiate and analyze individual cell populations. Here, we integrated a single-cell RNA sequencing profile of CML stem cells and network analysis to decipher the mechanisms of distinct TKI responses. Compared to normal hematopoietic stem cells, a set of genes that were concordantly differentially expressed in various types of stem cells of CML patients was revealed. Further transcription regulatory network analysis found that most of these genes were directly controlled by one or more transcript factors and the genes have more regulators in the cells of the patients who responded to the treatment. The molecular markers including a known drug-resistance gene and novel gene signatures for treatment response were also identified. Moreover, we combined protein-protein interaction network construction with a cancer drug database and uncovered the drugs that target the marker genes directly or indirectly via the protein interactions. The gene signatures and their interacted proteins identified by this work can be used for treatment response prediction and lead to new strategies for drug resistance monitoring and prevention. Our single-cell-based findings offered novel insights into the mechanisms underlying the therapeutic response of CML.

Hematopoietic and Chronic Myeloid Leukemia Stem Cells: Multi-Stability versus Lineage Restriction.

There is compelling evidence to support the view that the cell-of-origin for chronic myeloid leukemia is a hematopoietic stem cell. Unlike normal hematopoietic stem cells, the progeny of the leukemia stem cells are predominantly neutrophils during the disease chronic phase and there is a mild anemia. The hallmark oncogene for chronic myeloid leukemia is the BCR-ABLp210 fusion gene. Various studies have excluded a role for BCR-ABLp210 expression in maintaining the population of leukemia stem cells. Studies of BCR-ABLp210 expression in embryonal stem cells that were differentiated into hematopoietic stem cells and of the expression in transgenic mice have revealed that BCR-ABLp210 is able to veer hematopoietic stem and progenitor cells towards a myeloid fate. For the transgenic mice, global changes to the epigenetic landscape were observed. In chronic myeloid leukemia, the ability of the leukemia stem cells to choose from the many fates that are available to normal hematopoietic stem cells appears to be deregulated by BCR-ABLp210 and changes to the epigenome are also important. Even so, we still do not have a precise picture as to why neutrophils are abundantly produced in chronic myeloid leukemia.

Measurable residual disease in childhood B-cell acute lymphoblastic leukemia.

Measurable residual disease in childhood B-cell acute lymphoblastic leukemia.

Splenic red pulp macrophages provide a niche for CML stem cells and induce therapy resistance

Disease progression and relapse of chronic myeloid leukemia (CML) are caused by therapy resistant leukemia stem cells (LSCs), and cure relies on their eradication. The microenvironment in the bone marrow (BM) is known to contribute to LSC maintenance and resistance. Although leukemic infiltration of the spleen is a hallmark of CML, it is unknown whether spleen cells form a niche that maintains LSCs. Here, we demonstrate that LSCs preferentially accumulate in the spleen and contribute to disease progression. Spleen LSCs were located in the red pulp close to red pulp macrophages (RPM) in CML patients and in a murine CML model. Pharmacologic and genetic depletion of RPM reduced LSCs and decreased their cell cycling activity in the spleen. Gene expression analysis revealed enriched stemness and decreased myeloid lineage differentiation in spleen leukemic stem and progenitor cells (LSPCs). These results demonstrate that splenic RPM form a niche that maintains CML LSCs in a quiescent state, resulting in disease progression and resistance to therapy.

HDAC I/IIb selective inhibitor Purinostat Mesylate combined with GLS1 inhibition effectively eliminates CML stem cells.

Purinostat Mesylate (PM) is a novel highly selective and active HDAC I/IIb inhibitor, and the injectable formulation of PM (PMF) based on the compound prescription containing cyclodextrin completely can overcome PM's poor solubility and improves its stability and pharmacokinetic properties. Here, we showed that PM effectively repressed the survival of Ph+ leukemia cells and CD34+ leukemia cells from CML patients in vitro. In vivo studies demonstrated that PMF significantly prevented BCR-ABL(T315I) induced CML progression by restraining leukemia stem cells (LSCs), which are insensitive to chemotherapy and responsible for CML relapse. Mechanism studies revealed that targeting HDAC I/IIb repressed several important factors for LSCs survival including c-Myc, β-Catenin, E2f, Ezh2, Alox5, and mTOR, as well as interrupted some critical biologic processes. Additionally, PMF increased glutamate metabolism in LSCs by increasing GLS1. The combination of PMF and glutaminase inhibitor BPTES synergistically eradicated LSCs by altering multiple key proteins and signaling pathways which are critical for LSC survival and self-renewal. Overall, our findings represent a new therapeutic strategy for eliminating LSCs by targeting HDAC I/IIb and glutaminolysis, which potentially provides a guidance for PMF clinical trials in the future for TKI resistance CML patients.

Ex Vivo Expansion of Phenotypic and Transcriptomic Chronic Myeloid Leukemia Stem Cells

Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establishex vivoexpanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor β signaling pathways. Drug testing on expanded LSCs show that transforming growth factor β and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.

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.

Chronic myeloid leukemia: where do we stand, where can we go?

The introduction of BCR-ABL tyrosine kinase inhibitors to the treatment of chronic myeloid leukemia (CML) has significantly changed the long term therapy results. After an initial 12 months of therapy with tyrosine kinase inhibitor (TKI), a 3-log reduction of the BCR-ABL copies number on an international scale is possible in 22–46% of patients, depending on the TKI used. In TKI-responsive patients, long-term TKI treatment results are even better, with the BCR-ABL transcript level decreasing over time, even to the point of becoming undetectable. Therefore, an operational cure can be diagnosed in CML patients with an optimal response to 1 st -line TKI treatment, atherapy duration of longer than 5–8 years, and BCR-ABL transcript level below MR4.0–MR4.5 for a period of more than two years. The latter has been the basis of multiple concepts of permanent or periodic discontinuation of TKI treatment [treatment-free remission (TFR)]. Initial TKI discontinuation clinical trials resulted in satisfactory results, with a disease recurrence rate of c.40–60% after 2–3 years. The mechanism of disease recurrence was then studied , with detailed characterization of the CML stem cells (CML SCs) immunophenotype and the mechanisms of survival and self-renewal under TKI selective pressure. A better understanding of the biology of CML allowed the formulation of new therapy concepts of CML SCs eradication, and new criteria for successful TFR qualification.

P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA

Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients. Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase 2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months. Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR. Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04). Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods.

ACY-1215 suppresses the proliferation and induces apoptosis of chronic myeloid leukemia cells via the ROS/PTEN/Akt pathway.

Chronic myeloid leukemia (CML) is a hematological tumor marked by the bcr-abl fusion gene formed by t (9;22) (q34; q11), which translated into the BCR-ABL protein. Tyrosine kinase inhibitors (TKIs) have been widely used to cure CML patients. Nevertheless, the emergence of TKI resistance has become the problem to the outcome of CML patients. Histone deacetylase 6 (HDAC6), a kind of Hsp90α deacetylase, was detected to be overexpressed in chronic myeloid leukemia stem cells. Besides, the loss of HDAC6 enzymatic activity can result in the degradation of Hsp90α's client proteins, such as BCR-ABL, the oncoprotein of CML. Here, we explored the expression of HDAC6 and discovered that it was upregulated compared with control in CML. Then we explored the effect of Rocilinostat (ACY-1215), a specific HDAC6 inhibitor, on CML cells. Our results proved that ACY-1215 could induce apoptosis and cell cycle arrest in a ROS-dependent manner. Moreover, we detected a downregulation of the BCR-ABL signaling pathway in the ACY-1215 treatment group. Mechanistically, we noted that the upregulation of PTEN was induced after being treated by ACY-1215 and its downstream protein p-Akt was decreased. The Akt activator SC79 can partially reverse the influence of ACY-1215 on CML cells. Besides, our results also proved that ACY-1215 can synergize with imatinib to suppress chronic myeloid leukemia in vitro and in vivo. On the whole, our study revealed that HDAC6 is a possible therapeutic target in CML, and the combination therapy of TKI and HDAC6 inhibitor may improve the outcome of CML patients.

Loss of PRMT7 reprograms glycine metabolism to selectively eradicate leukemia stem cells in CML

Our group has reported previously on the role of various members of the protein arginine methyltransferase (PRMT) family, which are involved in epigenetic regulation, in the progression of leukemia. Here, we explored the role of PRMT7, given its unique function within the PRMT family, in the maintenance of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Genetic loss of Prmt7, and the development and testing of a small-molecule specific inhibitor of PRMT7, showed that targeting PRMT7 delayed leukemia development and impaired self-renewal of LSCs in a CML mouse model and in primary CML CD34+ cells from humans without affecting normal hematopoiesis. Mechanistically, loss of PRMT7 resulted in reduced expressions of glycine decarboxylase, leading to the reprograming of glycine metabolism to generate methylglyoxal, which is detrimental to LSCs. These findings link histone arginine methylation with glycine metabolism, while suggesting PRMT7 as a potential therapeutic target for the eradication of LSCs in CML.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.

Up-regulation of miR-144 Inhibits CML CD34+ Cell Proliferation Through Modulating TGF-β Signaling

Background: TGF-β signaling plays a key role in the performance of leukemic stem cells (LSCs). According to the evidence, any disturbance in the TGF beta pathway function not only poses negative effects on the development of BCR-ABL-induced chronic myeloid leukemia (CML) but also depletes CML stem cells. Hence, it can be argued that the aberrant acquisition of self-renewal characteristic because of the aberrant activation of TGF-β signaling results in the CML progression, which indicates the importance of inhibiting other targets besides BCR-ABL, such as TGF-β signaling, that can regulate self-renewal of the leukemic, which in turn translates into inhibited growth of BCR-ABL-positive CD34+ LSCs. Methods: The CD34+ cells were isolated from three CML patients with no history of treatments. The cells were cultured in a Stemspan H3000 medium. After being characterized by flow cytometry, the CML CD34+ cells were transfected with miR-144. The expression levels of miR-144, Smad4, and TGFβR2 genes were evaluated by RT-PCR. The impact of miR-144 overexpression on CML CD34+ cells was determined by the evaluation of cellular proliferation. Results: Overexpression of miR-144 resulted in the down-regulation of Smad4 and TGF-βR2 and decreased cell proliferation in CML CD34+ cells. Conclusions: This study suggests the down-regulation of miR-144 as a potential contributor to CML pathogenesis by activating the oncogenic TGF-β pathway. Hence, the proliferation capability of CD34+ CML cells, as a potential source of relapse in CML patients, can be inhibited by restoring miR-144 expression.