Chronic Myelogenous Leukemia Model Research Articles

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 h. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2 A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Interleukin-9 production by type 2 innate lymphoid cells induces Paneth cell metaplasia and small intestinal remodeling

Paneth cell metaplasia (PCM) typically arises in pre-existing gastrointestinal (GI) diseases; however, the mechanistic pathway that induces metaplasia and whether PCM is initiated exclusively by disorders intrinsic to the GI tract is not well known. Here, we describe the development of PCM in a murine model of chronic myelogenous leukemia (CML) that is driven by an inducible bcr-abl oncogene. Mechanistically, CML induces a proinflammatory state within the GI tract that results in the production of epithelial-derived IL-33. The binding of IL-33 to the decoy receptor ST2 leads to IL-9 production by type 2 innate lymphoid cells (ILC2) which is directly responsible for the induction of PCM in the colon and tissue remodeling in the small intestines, characterized by goblet and tuft cell hyperplasia along with expansion of mucosal mast cells. Thus, we demonstrate that an extra-intestinal disease can trigger an ILC2/IL-9 immune circuit, which induces PCM and regulates epithelial cell fate decisions in the GI tract.

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

Bioinformatic and Molecular Interaction Studies Uncover That CCA-1.1 AND PGV-1 Differentially Target Mitotic Regulatory Protein and Have a Synergistic Effect against Leukemia Cells

Numerous studies demonstrated CCA-1.1, the modified compound from PGV-1, inhibits tumor cells growth in breast and colorectal cancer. This time, we used bioinformatics and molecular interaction approaches to ascertain the potential CCA-1.1 activity target focusing on leukemia, along with the cytotoxic test in leukemia cells. Genomics data expression was collected through the COSMIC database by selecting gene sets from K562 cells as a model for chronic myelogenous leukemia (CML). We identified CCA-1.1 and PGV-1 predicted targets through SwissTargetPrediction. The overlapping genes between the CCA-1.1, PGV-1, and K562 cells were chosen for further analysis. We narrowed down the potential targets by using the list of genes involved in the cell cycle and mitosis collected through GeneCards. A molecular docking study was applied to determine the molecular interaction between CCA-1.1 or PGV-1 and the predicted protein target. We carried out a cytotoxic test using a trypan blue exclusion assay. We treated K562 cells with CCA-1.1 and PGV-1 in single and combination treatment to determine the half concentration of growth inhibitory (GI50) and combination index (CI) score. CCA-1.1 and PGV-1 shared similar predicted target genes in mitosis, and interestingly CCA-1.1 were mainly targeted in Aurora A (AURKA) in K562 cells with lower docking scores against the inhibitor in molecular docking analysis. Moreover, each compound exhibited an inhibitory effect similarly, and the co-treatment resulted in a synergistic effect in K562 cells. Collectively, we indicated that CCA-1.1 and PGV-1 possibly targeted mitosis in cell cycle progression, and along with their specific targets, led to their synergistic activity in CML. These findings should be validated through experimental studies to provide more pharmacological activities of CCA-1.1 to cure CML.

Spred1 deficit promotes treatment resistance and transformation of chronic phase CML.

Spred1 is highly expressed in normal hematopoietic stem cells (HSCs). Lack of Spred1 function has been associated with aberrant hematopoiesis and acute leukemias. In chronic myelogenous leukemia (CML), Spred1 is reduced in patients with accelerated phase (AP) or blast crisis (BC) CML, thereby suggesting that deficit of this protein may contribute to disease transformation. In fact, Spred1 knockout (KO) in SCLtTA/BCR-ABL CML mice either globally, or restricted to hematopoietic cells (i.e., HSCs) or to endothelial cells (ECs), led to transformation of chronic phase (CP) CML into AP/BC CML. Upon BCR-ABL induction, all three Spred1 KO CML models showed AP/BC features. However, compared with global Spred1 KO, the AP/BC phenotypes of HSC-Spred1 KO and EC-Spred1 KO CML models were attenuated, suggesting a concurrent contribution of Spred1 deficit in multiple compartments of the leukemic bone marrow niche to the CML transformation. Spred1 KO, regardless if occurred in HSCs or in ECs, increased miR-126 in LSKs (Lin-Sca-1+c-Kit+), a populationenriched in leukemic stem cells (LSCs), resulting in expansion of LSCs, likely through hyperactivation of the MAPK/ERK pathway that augmented Bcl-2 expression and stability. This ultimately led to enhancement of Bcl-2-dependent oxidative phosphorylation that supported homeostasis, survival and activity of LSCs and drove AP/BC transformation.

ANP32B-mediated repression of p53 contributes to maintenance of normal and CML stem cells

AbstractProper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell–specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and postinjury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with p53 and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from patients with chronic myelogenous leukemia (CML). Anp32b deletion enhances p53 transcriptional activity to impair LSC function in a murine CML model and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B and identify ANP32B as a potential therapeutic target in treating CML.

CCL3 Signaling Is Essential for Leukemia Progression but Dispensable for Hematopoietic Stem Cell Maintenance

Background: Hematologic malignancies remodel the bone marrow microenvironment (BMME), reducing support for normal hematopoiesis while increasing support for the malignancy. The chemokine CCL3 has been demonstrated to play a role in BMME dysfunction in multiple hematologic malignancies including myeloma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and myelodysplastic syndrome. Therefore we used murine models to analyze the requirement for CCL3 expression in leukemia progression and the viability of CCL3 signaling as a target in myelogenous leukemias.Methods/Results: We utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. We used sorted hematopoietic stem and progenitor cells (HSPC) (Lineage-sca1+ckit+) from CCL3KO mice or wild type controls to generate 2 models of AML, the BCR/ABL+Nup98/HoxA9 model of blast crisis CML (bcCML), and the MLL/AF9 model of AML. In the bcCML model, CCL3KO leukemic cells do not generate a leukemic disease when transplanted into naïve WT recipient mice in contrast to WT leukemic cells that will generate an acute myeloid leukemia with 100% penetrance over the course of 15-20 days. In the MLL/AF9 AML model, the disease progression of CCL3KO leukemic cells is significantly delayed as compared to WT controls (WT vs. CCL3KO p<0.05 n=15 mice/group). Therefore CCL3 modulates leukemogenesis in two genetically divergent AML models. The small molecule maraviroc is FDA approved and inhibits ligand binding to the chemokine receptor CCR5, one of the receptors for CCL3. Due to the poor pharmacokinetics of maraviroc in mice we utilized a novel micelle nanoparticle (NP) delivery platform that is targeted to the BMME through the use of a tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP). Using this platform we can delay the release of maraviroc over 48 hours, and target that release to the bone marrow. To verify that CCL3 signaling is targetable in hematologic malignancies, and that the BMME targeted NP platform is a viable method of drug delivery we loaded maraviroc into TBP-NPs and treated the murine model of bcCML every 2 days over the course of 10 days. The bcCML model was used for these studies due to its exquisite sensitivity to CCL3 expression. In bcCML mice the BMME is altered including an increase in phenotypic MSC populations and HSPC populations. Following treatment with maraviroc TBP-NPs the increase in MSC and HSPC populations is reversed and leukemic burden was decreased >2 fold in the bone marrow (VEH vs Maraviroc TBP-NP treated p<0.01 n=10 mice/group). These data suggest that CCL3 can play a critical role in the progression of myelogenous leukemias and is a potential target for therapy.If CCL3 signaling is to be a viable therapeutic target in AML it must be specific to leukemic cells while sparing the normal hematopoietic system. Therefore we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for long term repopulating HSPCs (LT-HSCs) in order to establish stem cell activity on a per cell basis through competitive transplantation. Upon secondary transplantation, the true test of LT-HSC function, CCL3KO Flt3-LSK donor cells engrafted in recipient mice at a higher rate (2-way ANOVA, p<0.05 over 16 weeks, n=8 mice/group). These results confirm that CCL3KO mice maintain a functional LT-HSC population.Conclusions: These results demonstrate that in multiple mouse models of myelogenous leukemia CCL3 plays an important, and in some cases, indispensable role in leukemogenesis, likely involving dysruption of the normal BMME. Importantly however, a long-term engrafting normal HSC population is clearly maintained even in the complete absence of CCL3 suggesting that anti-CCL3 therapy would be well-tolerated by the normal hematopoietic system. DisclosuresNo relevant conflicts of interest to declare.

Role of Signal Transducing Adaptor Protein (STAP) Family in Chronic Myelogenous Leukemia

Role of Signal Transducing Adaptor Protein (STAP) Family in Chronic Myelogenous Leukemia

Gata2-L359V impairs primitive and definitive hematopoiesis and blocks cell differentiation in murine chronic myelogenous leukemia model

GATA2, a key transcription factor in hematopoiesis, is frequently mutated in hematopoietic malignancies. How the GATA2 mutants contribute to hematopoiesis and malignant transformation remains largely unexplored. Here, we report that Gata2-L359V mutation impeded hematopoietic differentiation in murine embryonic and adult hematopoiesis and blocked murine chronic myeloid leukemia (CML) cell differentiation. We established a Gata2-L359V knockin mouse model in which the homozygous Gata2-L359V mutation caused major defects in primitive erythropoiesis with an accumulation of erythroid precursors and severe anemia, leading to embryonic lethality around E11.5. During adult life, the Gata2-L359V heterozygous mice exhibited a notable decrease in bone marrow (BM) recovery under stress induction with cytotoxic drug 5-fluorouracil. Using RNA sequencing, it was revealed that homozygous Gata2-L359V suppressed genes related to embryonic hematopoiesis in yolk sac, while heterozygous Gata2-L359V dysregulated genes related to cell cycle and proliferation in BM Lin-Sca1+c-kit+ cells. Furthermore, through chromatin immunoprecipitation sequencing and transactivation experiments, we found that this mutation enhanced the DNA-binding capacity and transcriptional activities of Gata2, which was likely associated with the altered expression of some essential genes during embryonic and adult hematopoiesis. In mice model harboring BCR/ABL, single-cell RNA-sequencing demonstrated that Gata2-L359V induced additional gene expression profile abnormalities and partially affected cell differentiation at the early stage of myelomonocytic lineage, evidenced by the increase of granulocyte–monocyte progenitors and monocytosis. Taken together, our study unveiled that Gata2-L359V mutation induces defective hematopoietic development and blocks the differentiation of CML cells.

Examination of Clinically-Derived p210 BCR/ABL Rhogef Mutations in a Murine Bone Marrow Transplantation Model of CML

The Philadelphia chromosome is formed by a balanced, reciprocal translocation that pairs sequences from BCR on Chromosome 22 with sequences from ABL on Chromosome 9, resulting in the production of the constitutively active tyrosine kinase (TK) BCR-ABL. Depending on the location of the breakpoint within BCR, three different sizes of BCR-ABL may form, each associated with distinct clinical sequelae. Previous studies identified a functional domain within the BCR sequences preserved by the more-indolent forms of BCR-ABL (i.e., p210 and p230) that demonstrates a constitutive Rho guanine nucleotide exchange factor (RhoGEF) activity. Using the structurally derived S509A mutation, which does not affect TK activity but abrogates RhoGEF signaling, we, subsequently, showed that this feature regulates leukemia progression in mice. The RhoGEF domain was recently reported to contain two missense mutations (F547L and T654K) in a chronic myelogenous leukemia (CML) blast crisis (BC) patient, suggesting that it may play a role in human disease as well. To evaluate the clinical significance of this region, we, therefore, examined p210 BCR-ABL constructs containing these clinically derived RhoGEF mutations (CDRMs), both in isolation and in combination, using a murine bone marrow transplant (mBMT) model of CML. The mutations did not destabilize p210 BCR-ABL expression or TK activity but decreased RhoGEF signaling. Relative to mice transplanted with wild-type (WT) p210 BCR-ABL, those that received the CDRMs exhibited an earlier onset of disease, frequently developing previously unseen dermatologic involvement or myeloid sarcomas, but demonstrated significantly increased survival in an additive manner [Fig. 1]. While mice transplanted with WT p210 BCR-ABL exhibited neutrophilia that progresses to a less-differentiated phenotype at death, disease in the CDRM mice was characterized by eosinophilia and monocytosis with no maturation arrest. The most likely cause of death in mice transplanted with WT p210 BCR-ABL is widespread hematogenous involvement of the lungs resulting in acute respiratory distress. In contrast, mice receiving the CDRMs demonstrated pulmonary involvement which was limited to the bronchovascular bundles or subpleural space. This is consistent with a switch to lymphatogenous spread, likely secondary to skewed differentiation, and it implies an alternate cause(s) of death. To help determine the mechanism responsible for the observed differences in differentiation and maturation arrest, we next studied the CDRMs in vitro using the 32Dcl3 murine myeloid precursor cell line. A growing body of evidence suggests that the DNA damage response can affect lineage restriction in myeloid cells; it is also well-established that the accumulation of DNA damage is necessary for progression to BC. Accordingly, the CDRMs were associated with the restoration of p53 activation and the G1/S cell cycle checkpoint relative to WT p210 BCR-ABL. This suggests that the RhoGEF domain may be responsible for the suppression of the DNA damage response. Because the mBMT phenotype associated with the S509A mutation was distinct from that of the CDRMs, it may indicate that this capability is mediated by some functionality yet to be described. Importantly, the CDRMs were also associated with increased Src family kinase activity, which is thought to be a major mechanism of BCR-ABL-independent TK inhibitor resistance and minimal residual disease. Collectively, these results suggest that the BCR-ABL RhoGEF domain can influence disease progression and response to treatment in human leukemia. Disclosures No relevant conflicts of interest to declare.

Examination of clinically-derived p210 BCR/ABL1 RhoGEF mutations in a murine bone marrow transplantation model of CML

Expression of the p210 BCR/ABL1 fusion protein has been described in virtually all patients with chronic myelogenous leukemia (CML). Previous studies have identified a guanine nucleotide exchange factor (RhoGEF) domain within BCR that is retained in p210 BCR/ABL1. Missense mutations at residues T654 (T654K) and F547 (F547L) within this domain have been reported in a CML patient in blast crisis (BC). In this study, we have evaluated p210 BCR/ABL1 constructs that contain these substitutions in a murine bone marrow transplantation (BMT) model of CML. The mutants exhibit normal expression and tyrosine kinase activity but altered signaling. When examined in the BMT assay, mice that express the mutants exhibit earlier onset of disease but have significantly extended lifespans relative to mice that express unmodified p210 BCR/ABL1. While mice that express p210 BCR/ABL1 exhibit neutrophilia that progresses to a less differentiated phenotype at death, disease in the mutant mice is characterized by eosinophilia with no maturation arrest. This observation was confirmed in vitro using myeloid cells and was associated with enhanced p53 phosphorylation and G1/S arrest. These results suggest that residues within the RhoGEF domain of p210 BCR/ABL1 can influence disease progression.

Low-Density Lipoproteins, High-Density Lipoproteins (HDL), and HDL-Associated Proteins Differentially Modulate Chronic Myelogenous Leukemia Cell Viability.

Cellular lipid metabolism, lipoprotein interactions, and liver X receptor (LXR) activation have been implicated in the pathophysiology and treatment of cancer, although findings vary across cancer models and by lipoprotein profiles. In this study, we investigated the effects of human-derived low-density lipoproteins (LDL), high-density lipoproteins (HDL), and HDL-associated proteins apolipoprotein A1 (apoA1) and serum amyloid A (SAA) on markers of viability, cholesterol flux, and differentiation in K562 cells-a bone marrow-derived, stem-like erythroleukemia cell model of chronic myelogenous leukemia (CML). We further evaluated whether lipoprotein-mediated effects were altered by concomitant LXR activation. We observed that LDL promoted higher K562 cell viability in a dose- and time-dependent manner and increased cellular cholesterol concentrations, while LXR activation by the agonist TO901317 ablated these effects. LXR activation in the presence of HDL, apoA1 and SAA-rich HDL suppressed K562 cell viability, while robustly inducing mRNA expression of ATP-binding cassette transporter A1 (ABCA1). HDL and its associated proteins additionally suppressed mRNA expression of anti-apoptotic B-cell lymphoma-extra large (BCL-xL), and the erythroid lineage marker 5'-aminolevulinate synthase 2 (ALAS2), while SAA-rich HDL induced mRNA expression of the megakaryocytic lineage marker integrin subunit alpha 2b (ITGA2B). Together, these findings suggest that lipoproteins and LXR may impact the viability and characteristics of CML cells.

The novel Leal-polynomials for the multi-expansive approximation of nonlinear differential equations

This work presents the novel Leal-polynomials (LP) for the approximation of nonlinear differential equations of different kind. The main characteristic of LPs is that they satisfy multiple expansion points and its derivatives as a mechanism to replicate behaviour of the nonlinear problem, giving more accuracy within the region of interest. Therefore, the main contribution of this work is that LP satisfies the successive derivatives in some specific points, resulting more accurate polynomials than Taylor expansion does for the same degree of their respective polynomials. Such characteristic makes of LPs a handy and powerful tool to approximate different kind of differential equations including: singular problems, initial condition and boundary-valued problems, equations with discontinuities, coupled differential equations, high-order equations, among others. Additionally, we show how the process to obtain the polynomials is straightforward and simple to implement; generating a compact, and easy to compute, expression. Even more, we present the process to approximate Gelfand's equation, an equation of an isothermal reaction, a model for chronic myelogenous leukemia, Thomas-Fermi equation, and a high order nonlinear differential equations with discontinuities getting, as result, accurate, fast and compact approximate solutions. In addition, we present the computational convergence and error studies for LPs resulting convergent polynomials and error tendency to zero as the order of LPs increases for all study cases. Finally, a study of CPU time shows that LPs require a few nano-seconds to be evaluated, which makes them suitable for intensive computing applications.

Numerical analysis of three-dimensional leukemia model’s equilibrium points

The results of numerical analysis of a mathematical model for chronic myelogenous leukemia (CML) equilibrium points quantity are presented. Considering a control example, the condition for the system without bistable equilibrium points was reached, so the system is supposed to have only those equilibrium points which is are asymptotically stable.

CD93-Signaling Regulates Self-Renewal and Proliferation of Chronic Myeloid Leukemia Stem Cells in Mice and Humans and Might be a Promising Target for Treatment

Background: The introduction of BCR/ABL-specific tyrosine kinase inhibitors (TKIs) more than two decades ago revolutionized chronic myelogenous leukemia (CML) therapy. The majority of CML patients treated with TKIs obtain durable cytogenetic and molecular responses. However, only a subgroup of these patients can successfully discontinue TKI therapy and maintain a treatment-free remission (Laneuville et al. 2011). TKI-resistant leukemia stem cells (LSCs) persist in the majority of patients at low levels over a prolonged period. These quiescent, self-renewing LSCs in the BM are the major cause of relapse after drug discontinuation (Holyoake et al, 2017). The selective elimination of LSCs requires the definition of unique signaling pathways that promote self-renewal of LSCs but not of normal HSCs. Based on the documented expression of CD93 on LSCs (Kinstrie et al, 2015), the aim of the present study was to investigate the role of the cell surface receptor CD93 in the regulation of self-renewal of human and murine CML LSCs and its contribution to disease development and progression. Methods and Results: We found CD93 expression on LSCs and leukemia progenitor cells but not on more differentiated leukemia granulocytes in a murine retroviral lineage-negative Sca-1+ c-kit+ (LSK) transduction/transplantation CML model. Next-generation sequencing analysis revealed that Cd93-/- LSCs have a silenced gene expression signature particularly in genes involved in the regulation of gene expression, stem cell maintenance and proliferation. Out of the 1120 genes differentially expressed between BL/6 and Cd93-/- LSCs, 1108 genes were down-regulated. In contrast, naïve BL/6 and Cd93-/- hematopoietic stem cells (HSCs) did not display a dysregulation in these pathways. Functionally, CD93-deficiency in LSCs resulted in impaired self-renewal, reduced LSC frequencies in vitro (at least by a factor of 100, P&amp;lt;0.001) and in the incompetence to induce and propagate CML in mice. To study whether CD93-signaling in LSCs relies on ligand-binding to the extracellular domain of CD93, we generated an extracellular domain deletion mutant of CD93 (mCd93intra). Comparable to transduction with full-length mCd93, the expression of Cd93intra restored colony formation of Cd93-/- LSCs in vitro, suggesting that the maintenance of LSC self-renewal is independent of ligand-binding to the extracellular domain of CD93. Furthermore, analysis of the sub-cellular localization of CD93 in CML cells using a lentiviral expression vector encoding for AcGFP1-N1-Cd93 demonstrated nuclear localization of the CD93 intracellular domain (ICD). SCY1 like pseudokinase 1 (SCYL1), a regulator of gene transcription, directly interacts with the highly charged juxta membrane domain of the cytoplasmic tail of CD93 (Bohlson et al, 2005). Silencing of Scyl1 significantly reduced colony formation of BL/6 but not Cd93-/- LSCs in vitro suggesting that the ICD of CD93 regulates gene transcription via Scyl1 in CML LSCs. To discover compounds that affect LSC function similarly as genetic CD93 blockade, we performed a compound screen using the FDA approved drug library V2. The antiemetic agent metoclopramide, which is widely used in clinical routine to reduce nausea in cancer patients, was one very promising candidate identified in the screen. Metoclopramide treatment reduced clonogenic potential of CD93-competent LSCs to comparable levels as CD93-deficient LSCs in vitro without further affecting colony formation of CD93-deficient LSCs. Analysis of LSCs from newly diagnosed CML patients similarly demonstrated that CD93-signaling induces the expression of genes associated with proliferation and stemness, resulting in an increased clonogenic potential in vitro. In addition, colony formation and re-plating capacity in semisolid cultures of human CD34+CD38- LSCs was significantly impaired by metoclopramide at a pharmacological concentration of 0.1mM compared to control treatment. Conclusions: Overall, these results indicate that CD93-siganling is an important regulator of stemness and proliferation of human and murine CML LSCs. Furthermore, this study identifies expression of CD93 by LSCs as promising novel target for the treatment of CML. Disclosures Baerlocher: Novartis: Research Funding.

Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia

Abstract Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib. Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML. Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity. Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML. Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.

Role of Signal Transducing Adaptor Protein-1 (STAP-1) in Chronic Myelogenous Leukemia Stem Cells

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder caused by hematopoietic stem cells expressing the BCR-ABL fusion oncoprotein, which constitutively activates multiple signal transduction pathways such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and Janus kinase/signal transducer and activator of transcription (JAK/STAT). Although tyrosine kinase inhibitor (TKI) therapy results in dramatic clinical success, studies have shown that TKIs are unable to eradicate leukemic stem cells (LSCs). Several key signaling molecules and pathways have been proposed to regulate the survival of CML LSCs in the presence of TKI; however, the details remain unclear. It is necessary to elucidate the mechanisms that maintain LSCs to better understand the pathogenesis of CML and develop new treatment approaches.The family of signal-transducing adaptor proteins (STAPs), which includes STAP-1 and STAP-2, has been implicated in various intracellular signaling pathways. In 2003, we cloned STAP-2 as a c-fms interacting protein and reported that STAP-2 binds to BCR-ABL and enhances activity, leading to the activation of downstream molecules such as ERK, STAT5, BCL-xL, and BCL2. STAP-1 was cloned as a c-kit interacting protein from a hematopoietic stem cell library, but it is unknown whether STAP-1 plays a role in CML. Given the structural homology between STAP-1 and STAP-2 and the hematopoietic expression of STAP-1, we hypothesized that STAP-1 might contribute to the leukemogenesis of CML.A STAP-1-deficient (KO) CML mouse model was developed. To generate this model, lineage (Lin)− Sca-1+ c-Kithigh (LSK) fraction isolated from bone marrow (BM) cells was infected with a retrovirus carrying BCR-ABL1 and GFP and subsequently transplanted into congeneric recipients. STAP-1 KO CML mice showed significantly longer survival than WT CML mice and displayed less severe splenomegaly and lung hemorrhages compared with WT mice. In recipient BM, absolute numbers of STAP-1 KO LSCs (GFP+ LSK cells) were significantly lower than WT LSCs. In the colony-forming assay, STAP-1 KO LSCs generated fewer colonies compared to WT LSCs. Using flow cytometric analysis, we found that STAP-1 KO LSCs had a higher apoptotic rate than WT LSCs. These findings suggest that the suppression of apoptosis induced by STAP-1 mediates longer survival of LSCs. To further understand the effects of STAP-1, we performed a gene expression analysis using RNA-sequence (RNA-seq) and compared WT and STAP-1 KO CML LSCs. When canonical pathways were analyzed with Ingenuity Pathway Analysis, various pathways associated with inflammatory cytokines were observed to be regulated in STAP-1 KO CML LSCs. Changes in mRNA expression, including that of SOS1, SOS2, FOXO3, FASLG, NFKB2, and BCL-xL, indicated that the PTEN signaling pathway, known to play a tumor suppressive role in CML, was significantly activated by STAP-1 KO (p=1.096E-3, activation Z-score=2.611). The pathway related to JAK/STAT signaling was also affected (p=2.04E-5, activation Z-score=-3.286). Downstream genes in the JAK/STAT signaling pathway, such as STAT5B and BCL-xL, were downregulated more than 2-fold in STAP-1 KO LSCs, suggesting that the deletion of STAP-1 inhibits the expression of STAT5-targeted anti-apoptotic protein and induced apoptosis of CML LSCs. To confirm the results of the RNA-seq experiment, an intracellular flow cytometric assay with CML Lin− cells was conducted. The frequency of cells positive for phosphorylated STAT5 was reduced for STAP-1 KO compared with that for WT. Quantitative PCR with CML LSCs confirmed the downregulation of BCL2 and BCL-xL, which are STAT5-targeted anti-apoptotic genes, in STAP-1 KO CML LSCs.In conclusion, we show that STAP-1 plays a crucial role in the maintenance of CML LSCs using a murine model of CML. STAP-1 deficiency results in the reduction of phosphorylated STAT5, downregulation of anti-apoptotic genes BCL-2 and BCL-xL, and induced apoptosis of CML LSCs. These findings suggest that STAP-1 and related signaling pathways could be potential therapeutic targets for CML LSCs. DisclosuresIchii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Fujimoto Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

The Chemokine CCL3 Regulates Myeloid Differentiation and Hematopoietic Stem Cell Numbers

The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3−/− mice were used to evaluate the role of CCL3 in regulating hematopoietic stem and progenitor cell (HSPC) populations. CCL3−/− mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.

Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) stem cells (LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here, we show that although miR-126 supports the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels are lower in CML LSCs as compared to normal long-term hematopoietic stem cells (LT-HSCs). Down-regulation of miR-126 levels in CML LSCs is due to phosphorylation of SPRED1 by BCR-ABL, leading to inhibition of the RAN/EXP-5/RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using CML mouse models with conditional miR-126 knock-out (KO) in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment causes an undesired increase in endogenous miR-126 levels, thereby enhancing LSC quiescence and persistence. miR-126 KO in LSCs and/or ECs, or treatment with a CpG-miR-126 inhibitor targeting miR-126 in both LSCs and ECs, enhances the in vivo anti-leukemic effects of TKI treatment and strongly diminishes LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in CML.

Prostaglandin E1 and Its Analog Misoprostol Inhibit Human CML Stem Cell Self-Renewal via EP4 Receptor Activation and Repression of AP-1

Effective treatment of chronic myelogenous leukemia (CML) largely depends on the eradication of CML leukemic stem cells (LSCs). We recently showed that CML LSCs depend on Tcf1 and Lef1 factors for self-renewal. Using a connectivity map, we identified prostaglandin E1 (PGE1) as a small molecule that partly elicited the gene expression changes in LSCs caused by Tcf1/Lef1 deficiency. Although it has little impact on normal hematopoiesis, we found that PGE1 treatment impaired the persistence and activity of LSCs in a pre-clinical murine CML model and a xenograft model of transplanted CML patient CD34+ stem/progenitor cells. Mechanistically, PGE1 acted on the EP4 receptor and repressed Fosb andFos AP-1 factors in a β-catenin-independent manner. Misoprostol, an FDA-approved EP4 agonist, conferred similar protection against CML. These findings suggest that activation of this PGE1-EP4 pathway specifically targets CML LSCs and that the combination of PGE1/misoprostol with conventional tyrosine-kinase inhibitors could provide effective therapy for CML.