Quiescent Leukemic Stem Cells Research Articles

Targeting Quiescent Leukemic Stem Cells in Acute Myeloid Leukemia

Targeting Quiescent Leukemic Stem Cells in Acute Myeloid Leukemia

SKF-96365 Expels Tyrosine Kinase Inhibitor-Treated CML Stem and Progenitor Cells from the HS27A Stromal Cell Niche in a RhoA-Dependent Mechanism.

A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment. Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment-a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche. This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway.

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease – with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Ferritinophagy Is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) poses significant challenges in terms of prognosis and treatment options. Leukemic stem cells (LSCs) contribute to therapy resistance, relapse and adverse outcomes. This study aimed to explore one of the major features of LSCs (e.g. quiescence) and its underlying molecular mechanisms, as well as identify potential targets for therapeutic intervention. Using in vivo cell labeling in patient-derived xenograft (PDX) assays, we identified a quiescent cell subpopulation with high LSC potential. Transcriptomics experiments conducted separately on quiescent and cycling cell subsets revealed gene signatures characteristic of quiescent (Q_AML_UP) and cycling cells (C_AML_UP). Using single-cell RNA sequencing, we identified clusters of quiescent leukemic cell that were highly enriched in gene signatures related to LSCs (Figure 1A). Through a machine learning approach, we identified a gene set highly specific of quiescent cells (named the QS35 gene signature) that emerged as a significant prognostic factor for poor outcomes in independent cohorts of AML patients (Figure 1B). Next, we aimed to identify new vulnerabilities of LSCs by investigating the quiescent subset of leukemic cells. Transcriptomics and proteomics approaches on quiescent vs cycling cells revealed enhanced autophagy within the quiescent subset. Additionally, the transferrin receptor TFRC and ferritin, two key regulators of iron metabolism, exhibited lower expression in the quiescent subpopulations. As intracellular iron availability is tightly regulated by ferritinophagy, a specific form of autophagy leading to the degradation of ferritin, we investigated the coordinated regulation of autophagy and iron metabolism in the quiescent LSC subset. Intriguingly, the intracellular iron pool was lower in quiescent LSCs, particularly in the CD34+CD38- compartment compared to more mature leukemic cells. Moreover, autophagy inhibition selectively induced cytotoxicity in the CD34+CD38- compartment and reduced the frequency of long-term culture-initiating cells (LTC-IC), and these effects were rescued by exogenous iron supply, underscoring the critical role of autophagy in iron bioavailability within the quiescent LSC subpopulation. To target ferritinophagy in this compartment, we depleted NCOA4, the main carrier in ferritinophagy responsible for delivering ferritin-bound iron to the autophagosome, using shRNAs in primary AML samples. Interestingly, NCOA4 was found more expressed in quiescent compared to cycling AML cells. In ex vivo assays, NCOA4 downregulation significantly reduced the frequency of LTC-IC and inhibited the clonogenic capacities of leukemic cells. Furthermore, NCOA4 invalidation abrogated leukemia initiation and self-renewal in serial transplantation experiments in vivo, particularly within the quiescent LSC population (Figure 2A). Additionally, treatment with a novel small molecule inhibitor named compound 9a that disrupts the NCOA4-ferritin interaction, depleted intracellular iron in primary AML cells, reduced their clonogenic potential and LTC-IC frequency ex vivo, and induced cell death specifically within the quiescent CD34+CD38- subset. Importantly, the viability of LSCs treated with compound 9a was rescued by the addition of iron, demonstrating that the cytotoxic effects of this compound were dependent on iron depletion. Notably, compound 9a exhibited minimal toxicity to normal hematopoietic cells ex vivo. In PDX models, in vivo short-term treatment with compound 9a decreased tumor burden and induced cell death within the CD34+CD38- LSC population (Figure 2B). Mechanistically, genetic of chemical inhibition of NCOA4 led to the accumulation of dysfunctional mitochondria, and to the generation of reactive oxygen species especially within the LSCs population. Furthermore, inhibition of NCOA4 by compound 9a decreased mitophagy, strongly suggesting that NCOA4 coordinates ferritinophagy and mitophagy pathways to increase intracellular iron, thereby specifically promoting the survival of LSCs. These findings highlight the critical vulnerability of quiescent LSCs to ferritinophagy. Thus, NCOA4 inhibition represents an innovative therapeutic approach for AML patients.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Intra-Leukemic IFNγ Signaling Mediates Cell Cycle Suppression and Chemoresistance in AML

Introduction: The acute myeloid leukemia stem cell (LSC) model proposes that LSCs are a quiescent and functionally distinct cellular fraction that can escape chemotherapy and produce disease during relapse. Here, we reevaluated the relationship between quiescence and LSCs, and explored cell non-autonomous mechanisms contributing to chemotherapy escape. Methods: Using the definition that LSCs can engraft in immunodeficient mice, we evaluated the serial engraftment capability and cell cycle status of xenotransplanted AML patient samples and AML cells derived from AML-iPSC (iAML). To functionally interrogate regulators of cell cycle and chemotherapy escape, we used molecular profiling and perturbation studies to identify and validate mechanisms of cell cycle suppression. Results: To test the paradigm that quiescent LSCs are responsible for relapse after chemotherapy, we xenotransplanted both G0 and cycling cells from fresh primary AML cells or iAML. Contrary to previous reports, both G0 and cycling cells were transplantable, indicating that LSCs can exist in any cell cycle status. Having demonstrated that cell cycle status is heterogeneous in primary AML and not linked to cell-autonomous LSC identity, we speculated that cell cycle can be regulated by cell-extrinsic mechanisms. In particular, we hypothesized that leukemia cells themselves can interact to affect cell cycle, engraftment, and response to chemotherapy. To investigate this hypothesis, we established primary patient co-injection xenograft models in which the co-injected samples could be identified using two different HLA antibodies (Figure). Each sample expanded when transplanted by itself. Surprisingly, in all three patient pair models, one of the two co-injected patient samples (“Suppressed”) displayed very limited and decreased engraftment relative to the other sample (“Dominant”). Importantly, the Suppressed samples displayed less active cycling in Co-injection mice than in Alone mice, suggesting that the presence of Dominant samples inhibited the cycling of Suppressed samples. However, when isolated from primary recipients for secondary transplantation, Suppressed samples from Co-injection and Alone mice demonstrated similar engraftment capacity and caused similar mortality. Similar results were obtained in an iAML syngeneic co-injection model, demonstrating that suppression also occurs in cells derived from the same patient. These findings support our hypothesis that AML cell cycle can be suppressed by other AML cells without affecting the Suppressed cells' ability to initiate disease. Next, to identify mechanisms regulating cell cycle suppression, we evaluated RNA-seq of Dominant and Suppressed samples from our AML co-injection xenograft models and single-cell RNA-seq data from 12 AML patient specimens described in the literature. Suppressed samples exhibited enrichment of IFNγ signaling, which was also observed in G1 cells compared to S/G2/M cells in patient samples, suggesting IFNγ involvement in regulating cell cycle. To validate that IFNγ signaling mediates suppression, we disrupted expression of the IFNγ receptor IFNGR1 using CRIPSR/Cas9 gene editing in the Suppressed samples in the co-injection model. IFNGR1 loss increased cell cycle in the Suppressed samples compared to controls. Thus, IFNγ signaling contributes to AML cell cycle suppression. Finally, we investigated the role of IFNγ signaling in modulating cell cycle and chemosensitivity by treating our co-injection model with chemotherapy with and without anti-IFNGR1 blocking antibody. RNA-seq of both Suppressed and Dominant samples before and after chemotherapy revealed that Dominant samples and samples that escape suppression after chemotherapy downregulate IFNγ receptor expression and signaling. Furthermore, in samples enriched for IFNγ signaling, blocking IFNGR1 before and during chemotherapy treatment increased cell cycling before chemotherapy and decreased leukemia burden after chemotherapy. Conclusion: Here, we show that AML cell cycle can be suppressed in a cell non-autonomous manner that is mediated by IFNγ signaling. Moreover, blocking IFNγ signaling can increase chemosensitivity through activating the cell cycle. These findings provide novel insights into the mechanisms of chemotherapy escape and highlight IFNγ signaling as a potential therapeutic target to counter chemoresistance.

A Phase 1b/2 Trial of Liposomal Cytarabine and Daunorubicin (CPX-351) in Intermediate to High-Risk Acute Myeloid Leukemia for Patients Who Have Failed an Initial Cycle of Induction Chemotherapy

Introduction Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with a high rate of recurrence following standard chemotherapy that continues to have a poor prognosis. While there have been numerous recent advances in the treatment of AML, seven days of continuous infusion cytarabine combined with three doses of an anthracycline medication (7+3) remains the most common backbone treatment for younger (<60) patients. Following 7+3 treatment, patients are typically assessed by a bone marrow biopsy 14-21 days after initial treatment, and 20%-30% of patients will have persistent disease at this time. This residual disease is typically treated with an additional round of the same induction treatment at a full or slightly reduced dose. Unfortunately, a complete response (CR) following re-induction is infrequent in intermediate and high-risk AML patients (CR rates of 35% and 18%, respectively). We have performed mass cytometry studies of AML cell cycle state and find that in patients with residual disease at day 14-21 of induction, leukemia stem and progenitor cells have a low proliferative fraction. We hypothesize that this low rate of proliferation reduces the effectiveness of re-induction therapy and could be countered using CPX-351, which is FDA-approved for treatment of therapy-related AML and AML with myelodysplasia-related changes. CPX-351 is a formulation of cytarabine and daunorubicin in a liposomal structure at an optimal 5:1 molar ratio that has been shown to be preferentially absorbed by hematopoietic cells and to persist in serum and bone marrow for significantly longer than traditional 7+3 chemotherapy. We hypothesize that the unique properties of CPX-351 will better target quiescent leukemia stem and progenitor cells and we thus designed a phase 1b/2 study to determine the safety and efficacy of CPX-351 as re-induction treatment for patients with intermediate and high-risk AML with residual disease after a first round of induction. Patients and Methods Eight patients with intermediate or high-risk AML who had failed to achieve hypocellular marrow 14-21 days following initial treatment with traditional 7+3 cytarabine and daunorubicin were treated with CPX-351 re-induction in the phase 1b portion of the study with a CPX-351 dose of daunorubicin 44 mg/m 2 and cytarabine 100 mg/m 2 given two or three times every 48 hours. Concomitant midostaurin (days 8-21) was allowed for patients with FLT3 mutations (Patients #1 and #8). Four patients were ELN intermediate-risk and four adverse-risk; mean blast count before re-induction was 45%. Patients were assessed weekly for signs of toxicity and for treatment response. Samples were collected from bone marrow biopsies performed before and after treatment for correlative mass cytometry studies using a 50-antibody panel to determine cell cycle state, intracellular signaling markers, and DNA damage response. Results Of the eight patients treated to date, six received two doses of CPX-351 and two received three doses. Of the six patients receiving two doses, five achieved a morphologic CR (two with residual genetic alterations), and one patient achieved a CRi. Two patients received three doses of CPX-351, with one achieving a PR and the other MLFS. Five of the eight patients were able to proceed to an allogeneic stem cell transplant. Consistent with previous studies of CPX-351, treatment was well-tolerated, almost all grade 3 or 4 toxicities were due to cytopenias (including two DLTs for delayed count recovery) and the only significant drug-related non-hematologic toxicity was a decrease in cardiac ejection fraction in patient #1. All treated patients survived >150 days and all were able to receive additional AML-directed therapy or HSCT after CPX-351 treatment. Due to slow accrual, hematologic toxicity, and the 100% CR/CRi rate in the two-dose group, the trial was amended to use two doses for the phase 2 portion, and an additional study site was opened. Study accrual is ongoing with a target of 20 additional patients in the phase 2 portion. Conclusion This ongoing phase 1b/2 trial of CPX-351 (liposomal cytarabine and daunorubicin) as re-induction treatment for intermediate and high-risk AML has been safe and tolerable with early treatment outcomes that appear to be much better than reported historical controls. Correlative studies by mass cytometry will further establish the mechanistic reasons for the observed responses.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p<0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells

AbstractTyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1–mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

Abstract A55: Role of ID2 in acute myeloid leukemia progression and resistance to chemotherapy

Abstract Acute myeloid leukemia (AML) is thought to be maintained by quiescent leukemia stem cells (LSCs), which are resistant to chemotherapy and responsible for relapse. Recent evidence suggests that a subset of LSCs with higher oxidative phosphorylation and/or increased senescence are responsible for resistance to chemotherapy and relapse. While several genes have been identified that are associated with the maintain LSC maintenance and promote resistance and relapse, the precious molecular mechanism(s) are currently not known. Inhibitor of DNA binding (ID) proteins are a group of Helix-Loop-Helix (HLH) transcription regulators, which function as dominant regulators of other HLH proteins (E proteins) that are required for normal hematopoietic, muscle, neuronal and other cell development. We found that loss of ID2 expression in murine hematopoietic cells results in activation, differentiation, and exhaustion of hematopoietic stem cells (HSCs). ID2 promotes quiescence by stabilizing HIF-1a expression. Analysis of TCGA data sets shows that high levels of ID2 expression is associated with reduced overall survival among AML patients, suggesting that ID2 may function to maintain LSCs. Similar to murine cells, knockdown (KD) of ID2 expression in human cord blood cells resulted in a loss of CD34+CD38− progenitors. Furthermore, knockdown of ID2 expression in the UT-7 AML cell line showed significantly reduced leukemogenic potential in NSG mice, suggesting that ID2 is required for AML cell growth in vivo. UT7-ID2-KD cell line shows significant reduction of colony number when cultured in low oxygen condition in vitro, suggesting a potential role of HIF protein. In comparison, overexpression (OE) of ID2 in the human AML cell line, MO7e, promotes their leukemic potential and burden in NSG mice in vivo, suggesting that ID2 may function to preserve LSC quiescence. Initial studies in primary AML-PDX cells showed reduced leukemic potential upon ID2 knock down. High level of ID2 expression is associated with poor chemotherapy response in patients. We found that MO7e-ID2-OE are resistance to Cytarabine (AraC), while UT7-ID2-KD are susceptible to AraC. Furthermore, we found that AML cell lines that survive AraC treatment show increased ID2 expression, suggesting that ID2 may promote survival of chemotherapy resistant AML cells. It has been reported that AraC resistance of AML cells is associated with increased senescence. We found that surviving UT7-ID2-KD cells after AraC treatment have significantly reduced senescence population compared to controls. Taken together, ID2 promotes normal murine and human HSC quiescence, and may function promote LSC maintenance and resistance to chemotherapy. Citation Format: Tanmoy Sarkar, Jennifer Lombardo, Shweta Singh, Kristbjorn O Gudmundsson, Holly M Morris, Shyam Saran, Jonathan R Keller. Role of ID2 in acute myeloid leukemia progression and resistance to chemotherapy [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A55.

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.

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.

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Role for PLK1 in Leukemia Stem Cell Function in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. While most patients achieve initial remission following standard chemotherapy, the majority will relapse and ultimately die. Poor outcomes in AML are tied to the properties of leukemia stem cells (LSC), which drive therapy resistance and relapse. For successful therapeutic targeting, it is critical to understand the pathways that operate in LSC function and survival and how they are impacted by drug activity. We carried out an innovative stemness-focused high-throughput drug screen employing a hierarchical AML cell model and a Nanostring-based LSC signature assay to probe for genes and pathways that impact LSC function. All 4 small molecule inhibitors of Polo-like Kinase 1 (PLK1) included in the compound library were among the top hits. PLK1 inhibitor treatment selectively targeted the CD34⁺CD38⁻ fraction of OCI-AML-8227 cells, which is enriched for quiescent LSCs. PLK1 is an important cell cycle regulator; although there are reports for cell cycle-independent roles, they have not been systematically characterized. Importantly, the role of PLK1 has not been previously studied in the context of cancer stem cells. To investigate the role of PLK1 in LSCs, we performed single-cell (sc) multi-omics incorporating both scRNAseq and scATACseq on the CD34⁺CD38⁻ compartment of OCI-AML-8227 cells. Enrichment analysis of previously-identified chromatin accessibility signatures of primitive long-term hematopoietic stem cells (LT-HSC) and progenitors was used to identify cell clusters with distinct stemness and differentiation properties. Interestingly, PLK1 mRNA expression in OCI-AML-8227 was restricted to cells enriched for the LT-HSC epigenetic signature (Fig 1A). In healthy human bone marrow, PLK1 expression was restricted to committed progenitors and not detected in HSCs (Fig 1B). These data strongly suggest a previously unexplored role of PLK1 in regulating stemness that may be specific LSCs and not normal HSCs. To determine the effects of PLK1 against LSCs in vivo, we carried out PLK1 inhibitor treatment using the specific PLK1 inhibitor volasertib in a cohort of 9 primary AML samples in PDX models. Volasertib treatment significantly reduced leukemic burden in 7 of 9 samples, all of which had a high LSC17 score (associated with poor prognosis and standard chemotherapy response). To quantify effects on LSCs, we serially transplanted 2 samples at limiting doses into untreated secondary recipients and demonstrated a 5- to 25-fold reduction in LSC frequency following volasertib treatment, indicating that functional LSCs were targeted by PLK1 inhibition. Together, these data strongly suggest that PLK1 impacts LSC function and stemness, and that PLK1 inhibitors are acting through an uncharacterized cell-cycle independent mechanism to target quiescent LSCs. To discover novel PLK1 signaling pathways, we carried out proximity-dependent biotinylation (PDB) to characterize the PLK1 interactome in serum-starved cells. Preliminary analysis has revealed a number of previously uncharacterized PLK1 interactors, including components of autophagy and apoptosis machinery, that are currently being validated in the hematopoietic cell context. Our study highlights PLK1 as a specific vulnerability of LSCs, and furthers the understanding of malignant PLK1 signaling in non-cell cycle related processes. Further insights into the underlying mechanisms will enable the development of effective therapies to target these pathways and improve patient outcomes in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Analyses: Safety and Efficacy of Gemtuzumab Ozogamicin and Venetoclax in Patients with Relapsed or Refractory CD33+ Acute Myeloid Leukemia: A Phase Ib/II Study

Background: Results of therapy for relapsed/refractory (R/R) AML patients (pts) remain poor, with few surviving >2 years. Refractoriness to treatment may be due to the resistance of quiescent leukemia stem cells (LSCs) to conventional chemotherapy. Gemtuzumab Ozogamicin (GO), a monoclonal antibody linked to the cytotoxin calicheamicin, targets CD33, an antigen with variable expression on LSC. However, decreased blast CD33 expression, p-glycoprotein upregulation and decreased mitochondrial apoptosis are recognized mechanism of resistance to GO (Linenberger et al Leukemia 2005; 19:176-82). GO-induced apoptosis requires Bax and Bak effectors and is opposed by the anti-apoptotic proteins BCL-2 and BCL-XL. Thus, Venetoclax (VEN), a BH3 mimetic with high affinity for BCL-2 should facilitate GO-mediated LSC apoptosis. In addition, LSC, due to their reliance on oxidative phosphorylation (Lagadinou, Cell Stem Cell 2013, 12:329-41) are also uniquely sensitive to BCL-2 inhibition. Thus, we hypothesized a potential synergy between these drugs in targeting LSC in R/R AML. Study design: Big Ten Cancer Research Consortium (BTCRC)-AML17-113 is a single arm, open-label, multi-center, dose-escalation phase Ib/II study of the combination of GO and escalating doses of VEN in R/R AML pts (18-75y), using a 3+3 design. Correlative studies include pre-treatment AML blast CD33 expression and BH3 profiling, minimal residual disease (MRD) measurements at post-therapy time points using digital drop PCR technology, and quality of life (QOL) assessments (EORTC QLQ-C30, FACT-Fatigue). Methods: The primary endpoint is to determine the maximum tolerated dose (MTD) of VEN with GO. Secondary endpoints include overall response rate (ORR), anti-leukemic activity, characterization of treatment emergent adverse events (TEAEs), and estimates of relapse free survival (RFS), event free survival (EFS), and overall survival (OS). Eligibility: ECOG 0-2, adequate organ function, CD33+ in ≥20% AML blasts, ≤2 lines of prior therapy, no prior use of GO, no history of veno-occlusive disease (VOD), no BMT within 2 months, no CNS disease or HIV. Induction: 3-day VEN ramp-up to the target dose of 200 (cohort #1), 400 (#2), or 600 (#3) mg daily x 28d, with GO 3mg/m2 infused d1, 4, and 7. Consolidation/Maintenance: If CR/CRi achieved, pts proceed to BMT if applicable. If no BMT, or if partial response (PR), pts are consolidated with VEN at the prescribed dose x 28d with GO 3mg/m2 given d1 and 4 (Cycle 2). If BMT not applicable, and in CR/CRi, VEN alone is then given as maintenance in cycles 3+ until disease progression or toxicity. Updated Analysis: Trial design was previously described by Arain, S et al. JCO 2020, 15:suppl. TPS7566. Cohort#3 is completing accrual with the last of 6 patients currently undergoing induction treatment. Enrollment n=12 pts. Barriers to accrual have been the COVID-19 pandemic and widespread use of VEN/HMA by referring community oncologists. The study has been amended to allow for prior use of VEN containing regimens. SAE: Respiratory failure gr 3 (n=1), febrile neutropenia (FN) gr 3/4 (n=3), Anemia/rectal pain gr 3/4 (n=1). TEAE:(%;max grade (gr)): thrombocytopenia and anemia (both 60%;gr 4); neutropenia (40%;gr 4); pruritus (30%;gr 3); FN (20%;gr 4), weight loss, diarrhea , headache, constipation (all 20%;gr 1-2). Outcomes: None of the 6 pts in cohorts #1 and #2 responded to treatment. One pt developed VOD gr 4, related to the use of total marrow irradiation conditioning regimen post-induction with GO/VEN. In cohort #3, 3 patients are in CR (one post-HSCT), 2 are undergoing induction and 1 has progressed. Conclusion: The updated analysis of this study reveals a well tolerated drug combination with low toxicities, demonstrating responses in cohort#3 (VEN 600 mg). We plan to expand accrual to this cohort adding 6-12 pts. This trial is registered at clinicaltrials.gov as NCT040768.

Five-Year Follow-up of the Phase I/II Discontinuation Trial of Imatinib after Pioglitazone (EDI-PIO) in Chronic Myeloid Leukemia Patients in Deep Molecular Response

Treatment-free remission (TFR) is successful in approximately 40-60% of CML patients who achieve a stable and deep molecular response. Relapses may occur due to the persistence of quiescent leukemic stem cells (LSC). Pioglitazone, a drug used for diabetes, is a PPAR gamma agonist and reduces STAT5 activity, and its downstream targets HIF2α and CITED2, key guardians of the quiescent LSC. Residual LSC can be gradually purged from bone marrow niches by pioglitazone. Objectives: to evaluate the efficacy and safety of pioglitazone used with imatinib before treatment discontinuation. Methods: EDI-PIO (from the Portuguese Estudo de Descontinuação de Imatinibe após Pioglitazona) is a prospective, single arm, phase I/II discontinuation trial. Inclusion criteria: CML in chronic phase, treated with IM for at least 3 years, with MR4.5 reported by the International Scale (IS) for 2 years. Patients received pioglitazone 30 mg/day orally for three months before IM discontinuation. After discontinuation, BCR-ABL levels were measured by real-time quantitative PCR monthly for 12 months, every two months in the second year, and then every three months. IM was re-initiated at molecular relapse (single sample with a value of >0.1% or two consecutive samples >0.01%). TFR was calculated from IM discontinuation until molecular relapse, progression, or death to causes related to CML. Overall survival (OS) was calculated from IM discontinuation until the last seen or death date by any cause. This trial is registered with Clinicaltrials.gov, NCT02852486. Results: Between Jun/2016 and Jan/2019, we evaluated 32 CML patients, with a median age of 54 years (29-77), treated with imatinib for a median time of 9.5 years (3-16). The median duration of MR4 and MR4.5 was 106 and 93 months, respectively. The cut-off date for this analysis was July 1st, 2022. One patient left the trial before imatinib discontinuation and was not analyzed for TFR. There were no grade 3 or 4 adverse events related to pioglitazone. The median follow-up of the 31 patients that discontinued IM was 61 months (37-69). As previously reported, 15/31(48%) patients presented symptoms related to withdrawal syndrome. Twelve patients presented molecular relapse after a median time of 5 months (2-30). Nine relapses in the first six months and three at 7, 13, and 30 months after stopping imatinib. All relapsed patients re-achieved MMR in a median time of 3 months (1.8-4.1). One patient developed an anal canal adenocarcinoma in the third year after discontinuation and was treated with surgery and chemotherapy. TFR was 71%, 67%, 61% and 61% at 6,12, 30 and 60 months (Figure 1A), respectively. 60-month OS was 95% (CI 95%: 85-100%)(Figure 1B). There were 5 cases of COVID-19 among the 19 patients in TFR (26%) and two suspects. Four cases were mild, and one patient in MR4.5 died due to severe COVID-19. Sokal score and MR4.5 duration were significant factors for prolonged TFR (P=0.032 and 0.012, respectively). Conclusions: the combination of pioglitazone and IM was feasible and safe, with TFR rates consistent with previous discontinuation trials. The 5-year long-term follow-up demonstrated durable and stable molecular responses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CML-313 Kinetics of CITED2 Gene Expression in Chronic Myeloid Leukemia Patients

In CML, BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes for example CITED2 gene. This gene is involved in regulating the proliferation and quiescence states of leukemic stem cells (LSCs). Recently, studies reported that adding pioglitazone (PPARγ agonist approved for T2DM treatment) to imatinib may decrease the transcription of both STAT5 and CITED2 gene, eliminate the quiescent LSCs (resist TKIs), and ameliorate the patients' response. The primary objective is to clarify the value of combining pioglitazone with imatinib to downregulate the CITED2 gene aiming to eradicate the LSCs in CML treatment. In the context of a clinical trial (approved by the Ethical Committee of the Faculty of Medicine Mansoura University), the study group patients (N=26) were treated with combination therapy after having their consent. The planned follow-up time is 2 years. Hematology unit, Oncology Center, Mansoura University. The eligibility criteria for study group patients were de novo, Philadelphia+ve CML (chronic phase) aged 18-60 years old. Those patients were treated with imatinib 400 mg and pioglitazone 15 mg once daily for 6 months. Pre- and post-treatment samples (after 6 months) were collected to assess the expression levels of the CITED2 gene. Responders continued on the same TKI and their sequential BCR/ABL Q-PCR was monitored. The study proposed that this combination therapy can put more patients into deeper and faster molecular response via eliminating the LSCs. The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. The BCR-ABL and CITED2 genes' pretreatment expression levels decreased significantly after 6 months of combined treatment (p<0.001 and p=0.005, respectively). Moreover, at 6 months 65.4% and 23% of the patients had response ≥ CCyR and ≥ MMR respectively. The most frequent complaints among patients were increased body weight and edema (p<0.001). Adding pioglitazone to imatinib improved the patients' response and downregulate the overexpressed CITED2 gene. A longer follow-up is planned and needed to confirm the safety and efficacy of the combination.