Primary Chronic Myeloid Leukemia Cells Research Articles

Nutrient-Sensitising Drug Repurposing Screen Identifies Lomerizine As a Mitochondrial Metabolism Inhibitor in Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), the persistence of leukaemic stem cells (LSCs) following first line treatment with tyrosine kinase inhibitors (TKIs) such as imatinib, can lead to disease relapse. We have previously demonstrated that therapy-resistant CML LSCs rely on oxidative phosphorylation (OXPHOS) for survival and that targeting mitochondrial metabolism sensitises CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy, or are associated with adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. To identify clinically applicable compounds that target cells dependent on mitochondrial metabolism, we used a nutrient-sensitised repurposing screening strategy. Upon replacement of glucose with galactose as the only sugar source in cell culture media, there is a shift in cellular metabolism towards OXPHOS (as cells are unable to effectively use galactose for glycolysis). Therefore, CML cells grown in galactose-containing media are more sensitive to inhibition of mitochondrial respiration than glucose-grown cells. Using this approach, we screened total of >1,100 unique and structurally diverse FDA-approved drugs in CML cells, cultured in the presence of 11mM glucose or 11mM galactose. This uncovered lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against OXPHOS-dependent CML cells. Subsequently, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and an increase in mitochondrial Ca2+ levels compared with normal cells. Moreover, transcriptional analysis revealed that Ca2+ channel genes such as voltage-gated CACNA1D and receptor-activated TRPC6 are significantly upregulated in CML LSCs (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs), which was not reverted following imatinib treatment. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML LSCs was dependent on CACNA1D and TRPC6 activity, and the increase in mitochondrial Ca2+ levels, which could be inhibited by lomerizine treatment. Notably, CRISPR-Cas9-mediated CACNA1D or TRPC6 knockout significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and decreased cellular oxygen consumption rate, indicative of reduction in OXPHOS. Similarly, while imatinib treatment did not affect intracellular Ca2+ levels in primary CML CD34+ cells, lomerizine treatment significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and mitochondrial respiration/OXPHOS. Applying liquid chromatography-mass spectrometry and isotope assisted metabolomics, we traced labelled nutrients (glucose and palmitate) in CD34+ cells. This revealed a significant reduction in activity of mitochondria-located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity in primary CML cells following lomerizine treatment. Of clinical relevance, using combination of cell surface markers, cell division trackers and functional assays, including long-term culture initiating cell (LTC-IC) assay, we demonstrate that lomerizine treatment, alone or in combination with imatinib, targeted CML LSCs when cultured in physiological medium in vitro, without affecting normal HSCs. Notably, combination treatment with imatinib and lomerizine reduced CML tumour burden, targeted CML LSCs and extended survival in xenotransplantation model of human CML. In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via CACNA1D and TRPC6 is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, an FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.

Restoration of Mir-185 in Combination with BCR-ABL Downregulation By Therapeutic Delivery of siRNA with Lipid Nanoparticle Carriers Targets Drug Resistant Leukemic Stem/Progenitor Cells

Background: Despite development of effective tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), TKI therapy is not curative as some patients develop drug resistance or are at risk of disease relapse. Importantly, CML leukemic stem cells (LSCs) remain largely unresponsive and represent a key population for sustaining disease and relapse. We previously demonstrated that miR-185 levels were significantly reduced in CD34 + stem/progenitor cells from TKI-nonresponders compared to TKI responders. Further, restoration of miR-185 levels in CML LSCs and their progenitors impaired their survival and sensitized them to TKI in vitro and in vivo. However, the therapeutic potential of downregulating BCR-ABL using siRNA in combination with miR-185 mimic treatment to target CML LSCs have yet to be elucidated. Objective: Downregulation of BCR-ABL using siRNA represents a promising therapeutic approach to inhibit its kinase and non-kinase functions to combat drug-resistant CML and overcome limitations of current therapy. Additionally, previous work suggests that restoring miR-185 levels may improve therapeutic outcomes from complimentary therapies in CML, particularly in combination with BCR-ABL inhibition. Therefore, we aim to investigate the therapeutic outcomes and mechanism of action of inhibiting BCR-ABL using siRNA in combination with miR-185 mimic treatment in drug-resistant CML. Methods: To assess the effects of BCR-ABL downregulation in combination with miR-185 restoration, we encapsulated siRNA targeting BCR-ABL and a miR-185 mimic using an optimized lipid nanoparticle (LNP) formulation. Biological effects and mechanistic changes of this novel RNA combination was evaluated using various CML cell lines expressing b2a2, b3a2, T315I mutated BCR-ABL and CD34 + stem/progenitor cells from non-responder patients. Results: A total of 7 siRNA sequences targeting various fusion regions of BCR-ABL were designed and tested to effectively target primitive CML cells. The highest performing BCR-ABL siRNA encapsulated showed significant knockdown in various CML models expressing b2a2, b3a2 and T315I mutated BCR-ABL (65-99% knockdown). After 48 hours of 0.5 μg/mL BCR-ABL siRNA LNP treatment, BCR-ABL protein levels and its direct downstream target p-CrkL are undetectable by immunoblotting. The robust downregulation of BCR-ABL results in a dose-dependent decrease in cell viability (down to 28% and 9.4% viable cells at a dose of 1 μg/mL) and increase in apoptotic cells (70% and 78% Annexin-V positive) in imatinib resistant K562 and T315I mutated cells, respectively after 72 hours. Similarly, encapsulated miR-185 mimic downregulates its main downstream target PAK6 transcript levels down to 50% in drug sensitive and drug-resistant cell lines and in TKI nonresponder CD34 + stem/progenitor cells. More importantly, our RNA LNP formulation demonstrated significant uptake (> 80% Cy5 positive) of fluorescent-labelled siRNA in TKI nonresponder CD34 + stem/progenitor cells and decreased BCR-ABL mRNA transcript levels by 60%. The combined BCR-ABL inhibition and miR-185 mimic treatment were determined to be highly synergistic when analysed using the Chou-Talalay (combination index less than 1), ZIP, Bliss and HSA methods (synergy scores larger than 10) in both drug-sensitive and drug-resistant CML cells. Furthermore, treatment of BCR-ABL siRNA and miR-185 using LNPs decreased the colony forming units of nonresponder patient cells by 50%. Interestingly, this effect was further enhanced when combined with TKIs, decreasing colony forming units to 20% compared to control after 14 days. Conclusion: We have uncovered a LNP formulation that is able to effectively deliver therapeutic BCR-ABL siRNA and miR-185 mimic into primitive CML cells. This combination treatment significantly reduces the levels of oncogenic BCR-ABL and PAK6, decreasing the viability clonogenic potential and increases apoptosis in CML cell line models and primary patient cells. Importantly, this combination strategy overcomes TKI resistance in CD34 + stem/progenitor cells suggesting a feasible and effective model for patients suffering from clinical resistance and relapse.

Arginine dependency is a therapeutically exploitable vulnerability in chronic myeloid leukaemic stem cells.

To fuel accelerated proliferation, leukaemic cells undergo metabolic deregulation, which can result in specific nutrient dependencies. Here, we perform an amino acid drop-out screen and apply pre-clinical models of chronic phase chronic myeloid leukaemia (CML) to identify arginine as a nutrient essential for primary human CML cells. Analysis of the Microarray Innovations in Leukaemia (MILE) dataset uncovers reduced ASS1 levels in CML compared to most other leukaemia types. Stable isotope tracing reveals repressed activity of all urea cycle enzymes in patient-derived CML CD34+ cells, rendering them arginine auxotrophic. Thus, arginine deprivation completely blocks proliferation of CML CD34+ cells and induces significantly higher levels of apoptosis when compared to arginine-deprived cell lines. Similarly, primary CML cells, but not normal CD34+ samples, are particularly sensitive to treatment with the arginine-depleting enzyme, BCT-100, which induces apoptosis and reduces clonogenicity. Moreover, BCT-100 is highly efficacious in a patient-derived xenograft model, causing > 90% reduction in the number of human leukaemic stem cells (LSCs). These findings indicate arginine depletion to be a promising and novel strategy to eradicate therapy resistant LSCs.

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

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

Expansions of tumor-reactive Vdelta1 gamma-delta T cells in newly diagnosed patients with chronic myeloid leukemia

Recent studies have underscored the importance of gamma-delta (γδ) T cells in mediating potent MHC-unrestricted cytotoxicity in numerous malignancies. Here, we analyzed Vδ1 and Vδ2 γδ T cell subsets in newly diagnosed chronic myeloid leukemia (CML) patients (n = 40) who had initiated tyrosine kinase inhibitor (TKI) therapy including imatinib (n = 22), nilotinib (n = 14) and dasatinib (n = 4). Patient peripheral blood samples were analyzed at diagnosis and monitored prospectively at 3, 6, 12 and 18 months post-TKI. γδ T cells isolated from healthy donors and CML patients were used against K562, LAMA-84 and KYO-1 cell lines and against primary CML cells in cytotoxicity assays. We found large expansions of Vδ1 and Vδ2 T cells in patients at diagnosis compared to age-matched healthy donors (n = 40) (p < 0.0001). The γδ T cell reconstitution in patients on imatinib and also on nilotinib showed significant reductions of Vδ1 T cell and Vδ2 T cell absolute counts at 3 months compared to diagnosis. Importantly, Vδ1 and Vδ2 T absolute cell counts remained at normal levels from 3 months throughout the follow-up. Next, we observed susceptibility to specific lysis of primary CML tumor cells by Vδ1 T cells from healthy donors. Furthermore, we determined inherent cytotoxic reactivity by autologous patients’ Vδ1 T lymphocytes against primary CML tumor cells. Finally, the TCR clonality profiles showed in CML patients mostly polyclonal repertoires regardless of the TKI. Our results provide further evidence into γδ T cell antileukemia immunity in CML that might be beneficial for long-term disease control and treatment outcome.

BRD4 degradation blocks expression of MYC and multiple forms of stem cell resistance in Ph+ chronic myeloid leukemia.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream‐effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+/CD38− leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I. The BRD4‐targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1‐resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast‐induced TKI resistance of CML LSC in a co‐culture system and to block interferon‐gamma‐induced upregulation of the checkpoint antigen PD‐L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.

Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells.We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML.Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis.Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition.We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%.Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. DisclosuresNo relevant conflicts of interest to declare.

Statins Enhance the Molecular Response in Chronic Myeloid Leukemia when Combined with Tyrosine Kinase Inhibitors.

Simple SummaryApproximately 50–60% of patients with chronic myeloid leukemia (CML) achieve a stable deep molecular response (DMR) after tyrosine kinase inhibitor (TKI) therapy. The achievement of DMR is a prerequisite for treatment-free remission. Repurposing statins is a straightforward strategy for enhancing molecular response in CML treatment. Second-generation TKIs have been reported to exhibit cardiovascular toxicity. Thus, statins have been widely prescribed for patients with CML undergoing second-generation TKI therapy for modifying cardiovascular risk factors, such as hyperlipidemia. Furthermore, the results of this study support the therapeutic benefit of the concomitant use of statins in TKI therapy for patients with CML. Additionally, the potential additive effects of statins and TKIs enhance the DMR rate in patients with CML, rendering these effects clinically relevant in these patients. In particular, this combination is a strong candidate for the achievement of DMR in patients with CML who have not achieved DMR with TKI therapy alone.Previous studies have suggested that statins can be repurposed for cancer treatment. However, the therapeutic efficacy of statins in chronic myeloid leukemia (CML) has not yet been demonstrated. In this study, we retrospectively evaluated the outcomes of 408 CML patients who underwent imatinib therapy. The deep molecular response rates in patients treated with the statin/TKI combination were significantly higher than those in patients treated with TKI alone (p = 0.0016). The statin/TKI combination exerted potent cytotoxic effects against wild-type and ABL1 mutant CML, BaF3, and K562/T315I mutant cells. Furthermore, the statin/TKI combination additively inhibited the colony-forming capacity of murine CML-KLS+ cells in vitro. In addition, we examined the additive growth-inhibitory effects of the statin/tyrosine kinase inhibitor (TKI) combination against CML patient-derived CD34+ cells. The growth-inhibitory effects of the statin/imatinib combination against CD34+/CML primary cells were higher than those against CD34+/Norm cells (p = 0.005), suggesting that the combination of rosuvastatin and imatinib exerted growth-inhibitory effects against CML CD34+ cells, but not against normal CD34+ cells. Furthermore, results from RNA sequencing of control and statin-treated cells suggested that statins inhibited c-Myc-mediated and hematopoietic cell differentiation pathways. Thus, statins can be potentially repurposed to improve treatment outcomes in CML patients when combined with TKI therapy.

New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

Pharmacologic targeting of the P-TEFb complex as a therapeutic strategy for chronic myeloid leukemia

BackgroundThe positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with differentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells.Materials and methodsmRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell differentiation and apoptosis. Cell transfection, immunofluorescence analysis and co-immunoprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo.ResultsWe reported that the anti-CML effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex. We also confirmed the relationship between CDK9 and erythroid differentiation via knockdown the expression of CDK9. For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apoptosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor effects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human CD45+ cells in spleens of mice. We also verified that wogonin exhibited anti-CML effects through modulating P-TEFb activity in vivo.ConclusionsOur study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment.8cwZMBLp1zasMdEPaNEt3gVideo

Chidamide, a Novel Histone Deacetylase Inhibitor, Combined with Imatinib Induces Synthetic Lethality in Drug-Resistant Chronic Myeloid Leukemia

Background and ObjectiveTyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC) remain formidable challenges for chronic myeloid leukemia (CML). The second-generation TKI have been shown to overcome Imatinib-resistance in CML, but limited effect in CML cells harboring the T315I mutation and leukemia stem cells (LSC). Histone deacetylase inhibitors (HDACi) have shown promise as a therapy for TKI-resistance cells and CML-LSC. Chidamide (CS055/HBI-8000), a novel orally active HDACi, has been approved for treatment of T-cell lymphomas in multiple clinical trials, whereas its effect on CML patients has not been characterized. Here we investigated the effects of Chidamide alone and in combination with Imatinib(IM), on isogenic TKI-sensitive and -resistant CML cell lines, as well as BC-CML stem/progenitor cells.Methods and ResultsWe chosen CML cell lines (KBM5 cells and KBM5-T315I cells) and primary cells from 7 BC-CML patients and 5 healthy volunteers as experimental subject. We found that treatment with Chidamide alone inhibited cell proliferation, induced cell cycle G0/G1 arrest and apoptosis in Bcr-Abl expressing KBM5 cells by FCM. Co-treatment with Chidamide and Imatinib synergistically killed TKI-resistant CML cell line KBM5-T315I, and caused more reduction in phosphorylation of Bcr-Abl, STAT5 and CRKL by Western-blot, as compared with treatment with either agent alone (P <.05). Additionally, β-catenin is overexpressed in BC-CML stem/progenitor cells and can be induced by BM stromal cells, suggesting that β-catenin contributes to intrinsic and extrinsic Bcr-Abl kinase-independent TKI-resistance in CML. The co-treatment was also associated with a greater decrease in the protein levels of β-catenin, C-myc and Survivin, increase in the acetylation of Histone H3 and activation of Caspase-3/-9. Importantly, combination of Chidamide and Imatinib significantly down-regulated Bcr-Abl levels and induced apoptosis of CD34+ leukemia stem/progenitor cells derived from BC-CML patients with or without Bcr-Abl kinase mutations, but exhibited minimal toxicity to normal CD34+ progenitor cells.ConclusionCombined treatment with Chidamide and Imatinib exerts a superior anti-leukemia activity on CML cells including T315I mutation-harboring cell line, as well as BC-CML stem/progenitor cells. Our findings suggest potential benefit of the combination to overcome Bcr-Abl kinase-dependent and -independent TKI-resistance in CML, which may be a promising strategy to TKI-resistant BC-CML patients. DisclosuresNo relevant conflicts of interest to declare.

Delayed Expansion of Murine BCR-ABL1+ Leukemia By Genetic and Pharmacological Inhibition of NOX2

Reactive oxygen species (ROS), produced from reduction of oxygen by the myeloid NADPH oxidase (NOX2), are pivotal effector molecules in the innate immune defense against microbes, but imbalance between the generation and detoxification of ROS may be injurious to cell components. The malignant cells of patients with BCR-ABL1+ chronic myeloid leukemia (CML) show elevated ROS production, which has been implicated in the genetic instability BCR-ABL1+ cells. However, further studies are required to define the role of ROS in leukemogenesis. We have utilized genetically modified murine hematopoietic cells to determine the influence of NOX2-derived ROS for the in vivo expansion of transplanted BCR-ABL1+ cells. Irradiated C57BL/6J mice received transplants of wild-type (WT) or Nox2 knockout (Nox2 -KO) hematopoietic cells that were transduced to express GFP-labeled human BCR-ABL1 by use of the p210 construct cloned into a MSCV vector. In vitro experiments using the NOX2 inducer D-peptide confirmed that Nox2 -KO BCR-ABL1+ cells were devoid of ROS production via NOX2. Peripheral blood was drawn biweekly from mice receiving WT and Nox2 -KO transplants for fluorimetric quantification of BCR-ABL1+ cells. We observed prolonged survival for mice receiving Nox2 -KO vs. WT BCR-ABL1 transplants (n=43, p=0.017, logrank test) along with a trend towards reduced expansion of leukemic cells in vivo . To determine effects of pharmacological inhibition of NOX2 on leukemic expansion, the NOX2 inhibitor histamine dihydrochloride (HDC) was administered to mice carrying WT or Nox2 -KO BCR-ABL1+ transplants. In vitro experiments confirmed that HDC reduced NOX2-dependent induction of ROS formation in WT BCR-ABL1+ cells. Treatment of age-matched mice with HDC (1.5 mg three times per week i.p.) delayed the expansion of WT BCR-ABL1+ cells at four weeks after transplantation (n=18, p<0.05, t-test) but did not reduce the burden of leukemia in mice receiving Nox2 -KO BCR-ABL1+ transplants. Initial experiments using primary human CML cells revealed differential expression of redox-related genes in CD34+CD38- leukemic stem cells vs. cord blood stem cells. Our results implicate NOX2 as a conceivable therapeutic target in BCR-ABL1+ leukemia. DisclosuresGrauers Wiktorin:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

Plasma-Derived 3D Culture for Leukemic Proliferation

INTRODUCTIONAcute (AML) and chronic myeloid leukemia (CML) are hematological malignancies that remain incurable despite novel treatments. In order to improve current treatment options as well as clinical efficacy, there remains a need for more complex in vitro models that mimic the intricate human leukemic microenvironment. A bone marrow-derived 3D tissue engineered bone marrow (3DTEBM) model was previously developed to mimic the multiple myeloma pathophysiology, which allowed for patient material survival after one week in culture and recreated drug resistance. This study aimed to use 3D cultures derived from the peripheral blood of AML and CML patients to promote proliferation of leukemic cells and to be used as a drug screening tool for leukemic treatment. METHODS3D cultures derived from AML and CML patients were developed through calcium cross-linking of plasma from peripheral blood samples of AML or CML patients. AML cell lines (K052 and NOMO-1) and CML cell lines (K562 and Ku812) were incorporated in plasma-derived 3D cultures or 2D cultures in co-culture with stromal cell line Hs5, and tested for cell proliferation for 7 days by flow cytometry. In addition, five AML patient samples and five CML patient samples were incorporated in plasma-derived 3D cultures or 2D cultures and tested for cell proliferation for 7 days by flow cytometry. After optimization of cell growth, we tested the sensitivity of AML and CML cell lines to different chemotherapeutic agents, including alisertib (0 - 200 nM) and Ara-C (0 - 200 nM) for AML, and nilotinib (0 - 10 nM) and Cpd22 (0 - 1000 nM) for CML in plasma-derived 3D cultures or 2D cultures in co-culture with stromal cell line Hs5. RESULTSWe found that AML and CML cell lines demonstrated cell expansion rates of 2 and 2.5 fold and 2.5 and 3.5 fold in 2D cultures for days 3 and 7 compared to day 0, respectively. In contrast, AML and CML cell lines demonstrated higher cell expansion rates of 3.5 and 5 fold and 3.5 and 6 fold in plasma-derived 3D cultures for days 3 and 7 compared to day 0, respectively. In addition, while AML and CML patient samples showed non or very low cell expansion rates (lower than 1.5 fold) after 7 days in 2D cultures compared to day 0, AML and CML patient samples demonstrated cell expansion rates of 3 and 4.5 fold and 4 and 7.5 fold in plasma-derived 3D cultures for days 3 and 7 compared to day 0, respectively.Furthermore, we tested the effect of the 3D cultures on drug resistance of AML and CML cells. While Ara-C induced IC50 at about 100 nM in both AML cell lines in 2D cultures, only 25-35% of AML cells were killed at the same drug concentration. Alisertib induced IC50 at 100 nM in both AML cell lines in 2D cultures. However, at the same drug concentration only 5-10 % AML cells were killed in plasma-derived 3D cultures. Similarly, while in 2D co-cultures about 50-80% of CML cells were killed using the different concentrations of nilotinib and Cdp22, CML cell lines cultured in plasma-derived 3D cultures showed a significant decrease in sensitivity to the treatments with a 2-fold decrease in the number of cells killed. CONCLUSIONS3D tissue engineered AML and CML-derived cultures supported the growth of the primary AML and CML cells better than classic 2D systems. In addition, it induced significantly more drug resistance in AML and CML cells, compared to classic 2D culture systems. The 3D tissue engineered AML and CML cultures created a more physiologically relevant environment for leukemia cell proliferation, due to recreation of microenvironment cues including interactions with accessory stromal cells, and provided a reliable model for growing myeloid leukemia patient samples, serving as a relevant tool for drug screening, which could improve patient specific drug resistance testing for development of personalized medicine strategies in individual patients. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Azab:Targeted Therapeutics LLC: Other: Founder and owner; Verastem: Research Funding; Glycomimetics: Research Funding; Cleave Bioscience: Research Funding; Selexys: Research Funding; Cellatrix LLC: Other: Founder and owner; Cell Works: Research Funding; Tioma: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding.

3D tissue engineered plasma cultures support leukemic proliferation and induces drug resistance

Chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) are hematological malignancies that remain incurable despite novel treatments. In order to improve current treatments and clinical efficacy, there remains a need for more complex in vitro models that mimic the intricate human leukemic microenvironment. This study aimed to use 3D tissue engineered plasma cultures (3DTEPC) derived from CML, AML and CLL patients to promote proliferation of leukemic cells for use as a drug screening tool for treatment. 3DTEPC supported the growth of primary CML, AML and CLL cells and also induced significantly more drug resistance in CML, AML and CLL cell lines compared to 2D. The 3DTEPC created a more physiologically relevant environment for leukemia cell proliferation, provided a reliable model for growing leukemia patient samples, and serves as a relevant tool for drug screening and personalized medicine.

Inhibition of cystathionine \u03b2-synthase promotes apoptosis and reduces cell proliferation in chronic myeloid leukemia

Increased endogenous hydrogen sulfide (H2S) level by cystathionine β-synthase (CBS) has been shown to closely relate tumorigenesis. H2S promotes angiogenesis, stimulates bioenergy metabolism and inhibits selective phosphatases. However, the role of CBS and H2S in chronic myeloid leukemia (CML) remains elusive. In this study, we found that CBS and H2S levels were increased in the bone marrow mononuclear cells of pediatric CML patients, as well as in the CML-derived K562 cells and CBS expression levels were correlated with different disease phases. Inhibition of CBS reduced the proliferation of the CML primary bone marrow mononuclear cells and induced growth inhibition, apoptosis, cell cycle arrest, and migration suppression in K562 cells and tumor xenografts. The knockdown of CBS expression by shRNA and inhibiting CBS activity by AOAA decreased the endogenous H2S levels, promoted mitochondrial-related apoptosis and inhibited the NF-κB-mediated gene expression. Our study suggests that inhibition of CBS induces cell apoptosis, as well as limits cell proliferation and migration, a potential target for the treatment of chronic myeloid leukemia.

NT157, an IGF1R-IRS1/2 inhibitor, exhibits antineoplastic effects in pre-clinical models of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

Targeting Chronic Myeloid Leukemia Stem/Progenitor Cells Using Immunolipsome Loaded Venetoclax

The introduction of different generations of tyrosine kinase inhibitors (TKIs) significantly improved outcome and survival rate in chronic myeloid leukemia (CML) patients. However, long-term use of TKIs is concomitant with many side effects that affect the quality of life in patients. Approximately half of CML patients achieve deep molecular response (DMR), this makes them suitable candidates to discontinue the TKI therapy in a controlled condition, and about half of them will remain in treatment free remission (TFR) after discontinuation. It has been shown that a small population of leukemia stem cells (LSCs) as the residual disease burden is present at diagnosis, during the treatment, and in patients who are in TFR. While CML LSCs have many features in common with HSCs, they express specific markers such as CD25, CD26, IL1-RAP, etc., which can be used for the diagnosis and targeting. Protection by the bone marrow microenvironment and activity of signaling pathways such as WNT/β catenin, Hedgehog, PI3K, JAK/STAT in CML LSCs in a BCR-ABL dependent and independent manner guarantee their survival and elimination of these cells solely using TKIs seems ineffective. Herein we designed a pegylated liposomal nanocarrier conjugated with a specific antibody against CD26 (Begelomab, ADIENNE, Lugano, Switzerland). Then we loaded this immunoliposome with venetoclax, a BCL2 inhibitor, to eliminate CML LSCs selectively and to spare normal HSCs. First, we measured the expression of CD26 in the bone marrow and peripheral blood samples of newly diagnosed patients. We had a high expression of CD26 in CD34+/CD38- of both PB and BM, and a low expression on CD34+/CD38+ (progenitors) cells. Also, the expression of this marker in resistant patients to TKIs was visible while it was absent in normal stem cells. After the synthesis of the liposome, we conjugated Begelomab to the liposome. Then, we tested the selectivity of the designed system in different positive and negative cells. Our designed immunoliposome showed a strong selectivity toward CD26 positive cells. We also tested the selectivity on CML primary cells; in particular, we sorted newly diagnosed CML samples based on CD34+/CD38-/CD26- for HSCs and CD34+/CD38-/CD26+ for LSCs. Based on the confocal and flow cytometry analysis, our designed immunoliposome selectively targets LSCs and spares HSCs. Then we loaded this immunoliposome with venetoclax, and we treated CD26 positive and negative cells with this system. Based on our preliminary results, this immunoliposome loaded venetoclax specifically induced apoptosis in CD26+ cells, with higher activity compared to free venetoclax at the same dose. However, more analysis will be performed to confirm the selectivity of this system. Based on the obtained results, CD26 in newly diagnosed CML patients is expressed by CML LSCs and is a suitable option for diagnosis and targeting. Our preliminary data strongly suggest that we can selectively target CML LSCs. The main advantage of this system is its precision to hit the target. So we expect that after the drug release, the LSCs will be eliminated without any side effects on normal cells. Liposomes are suitable carriers because of their biocompatibility, self-assembly, large drug payload, and minimal toxicity. This strategy may help us to increase the number of patients attaining and maintaining TFR without relapsing. Disclosures Saglio: Ariad: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Roche: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding.

In Vitro Comparison of the Effects of Imatinib and Ponatinib on Chronic Myeloid Leukemia Progenitor/Stem Cell Features

BackgroundThe development of molecularly tailored therapeutic agents such as the BCR/ABL-active tyrosine kinase inhibitors (TKi) resulted in an excellent treatment option for chronic myeloid leukemia (CML) patients. However, following TKi discontinuation, disease relapses in 40–60% of patients, an occurrence very likely due to the persistence of leukemic stem cells that are scarcely sensitive to TKi. Nevertheless, TKi are still the only current treatment option for CML patients.ObjectiveThe aim of this study was to compare the effects of TKi belonging to different generations, imatinib and ponatinib (first and third generation, respectively), on progenitor/stem cell expansion potential and markers.Patients and MethodsWe used stabilized CML cell lines (KCL22, K562 and LAMA-84 cells), taking advantage of the previous demonstration of ours that cell lines contain cell subsets endowed with progenitor/stem cell properties. Primary cells explanted from CML patients were also used. The effects of TKi on the expression of stem cell related genes were compared by quantitative PCR. Flow cytometry was performed to evaluate aldehyde-dehydrogenase (ALDH) activity and the expression of cluster of differentiation (CD) cell surface hematopoietic stem cell markers. Progenitor/stem cell potential was estimated by serial colony formation ability (CFA) assay.ResultsPonatinib was more effective than imatinib for the reduction of cells with ALDH activity and progenitor/stem cell potential of CML patient-derived cells and cell lines. Furthermore, ponatinib was more effective than imatinib in reducing the percentage of CD26-expressing cells in primary CML cells, whereas imatinib and ponatinib showed similar efficacy on KCL22 cells. Both drugs strongly upregulated NANOG and SOX2 in CML cell lines, but in KCL22 cells this upregulation was significantly lower with ponatinib than with imatinib, an outcome compatible with a lower level of enrichment of the stem cell compartment upon ponatinib treatment.ConclusionPonatinib seems to target CML progenitor/stem cells better than imatinib.Electronic supplementary materialThe online version of this article (10.1007/s11523-020-00741-x) contains supplementary material, which is available to authorized users.

HERG K+ Channels Promote Survival of Irradiated Leukemia Cells

Many tumor cells express highly elevated activities of voltage-gated K+ channels in the plasma membrane which are indispensable for tumor growth. To test for K+ channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0–8 Gy, 6 MV photons) and determined K+ channel activity, K+ channel-dependent Ca2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K+ channels which became apparent 1–2 h post-irradiation. This increase in K+ channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G2/M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K+ channel function by applying the hERG1 channel inhibitor E4031 modulated Ca2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K+ channels for the Ca2+ signaling and the cell cycle control during DNA damage response.

CDK4/CDK6 inhibition as a novel strategy to suppress the growth and survival of BCR-ABL1T315I+ clones in TKI-resistant CML.

PurposePonatinib is the only approved tyrosine kinase inhibitor (TKI) suppressing BCR-ABL1T315I-mutated cells in chronic myeloid leukemia (CML). However, due to side effects and resistance, BCR-ABL1T315I-mutated CML remains a clinical challenge. Hydroxyurea (HU) has been used for cytoreduction in CML for decades. We found that HU suppresses or even eliminates BCR-ABL1T315I+ sub-clones in heavily pretreated CML patients. Based on this observation, we investigated the effects of HU on TKI-resistant CML cells in vitro.MethodsViability, apoptosis and proliferation of drug-exposed primary CML cells and BCR-ABL1+ cell lines were examined by flow cytometry and 3H-thymidine-uptake. Expression of drug targets was analyzed by qPCR and Western blotting.FindingsHU was more effective in inhibiting the proliferation of leukemic cells harboring BCR-ABL1T315I or T315I-including compound-mutations compared to cells expressing wildtype BCR-ABL1. Moreover, HU synergized with ponatinib and ABL001 in inducing growth inhibition in CML cells. Furthermore, HU blocked cell cycle progression in leukemic cells, which was accompanied by decreased expression of CDK4 and CDK6. Palbociclib, a more specific CDK4/CDK6-inhibitor, was also found to suppress proliferation in primary CML cells and to synergize with ponatinib in producing growth inhibition in BCR-ABL1T315I+ cells, suggesting that suppression of CDK4/CDK6 may be a promising concept to overcome BCR-ABL1T315I-associated TKI resistance.InterpretationHU and the CDK4/CDK6-blocker palbociclib inhibit growth of CML clones expressing BCR-ABL1T315I or complex T315I-including compound-mutations. Clinical studies are required to confirm single drug effects and the efficacy of `ponatinib+HU´ and ´ponatinib+palbociclib´ combinations in advanced CML.FundingThis project was supported by the Austrian Science Funds (FWF) projects F4701-B20, F4704-B20 and P30625.