Chronic Myeloid Leukemia Model Research Articles

Abstract 187: Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia

Abstract BCR-ABL1 fusion gene is the hall marker of chronic myeloid leukemia (CML). Currently, the immune microenvironment of BCRABL1 CML remains largely unexplored. Our recent investigation in a transgenic mouse model of BCRABL1 CML revealed a continuous increase in CD11b+Ly6Cint PMN-MDSC with the progression of the disease. Concurrently, there was an increase in CD11b+Ly6Chigh M-MDSC and F4/80+ macrophages. However, The levels of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD49b+ NK cells, which are essential for tumor-killing significantly decreased. This immune phenotype was consistently observed in both primary leukemia induction and following serial transplantation of cells from primary leukemic mice. Intriguingly, only recipient mice subjected to sublethal irradiation developed the disease, while mice with intact immune system remained disease-free, which underscores the vital role of the immune system in the development of CML. The predominant presence of CD11b+Ly6Cint PMN-MDSC in the peripheral blood, bone marrow and spleen implies these cells may be at least partially consisted of CML leukemia cells. To examine the hypothesis, qRT-PCR and immunofluorescence (IF) staining were conducted. In both assays, Ly6G+ cells from BCRABL1 mice coexpressed the BCRABL1 oncogene. Furthermore, when co-cultured with CD4 T cells, the division of T cells was significantly inhibited, implying immune suppression by the Ly6G+ PMN-MDSCs and/or CML leukemia cells. In addition, the immuosuppressive pathway genes including Arg1, Hif1a, Enptd1, TGFβ were significantly upregulated. To investigate the possible role of the PD1/PDL1 axis in the BCRABL1 model, flow cytometry analysis was conducted. Unexpectedly, PDL1 was barely expressed in CML leukemia cells, whereas the expression of PDL1 in M-MDSCs and macrophages significantly increased, accompanied by remarkable upregulation of PD1 expression on CD4 T cells and CD8 T cells during leukemia progression. In other words, PDL1 expression on macrophages, rather than leukemia cells, was found to induce T cell exhaustion. For translational purpose, mice transplanted with the primary CML leukemia cells were treated with chemotherapy drugs ponatinib alone or in combination with the anti-PD1 antibody. Compared with the chemotherapy alone group, the mice in the combinational therapy group exhibited rapid remission and better disease control. Most importantly, the disease relapse occurred at a slower rate after treatment discontinuation, suggesting the value of combinational therapy in achieving treatment free remission. In summary, our study not only revealed the dynamic changes of immune microenvironment in the BCRABL1 CML mouse model, but also revealed the different mechanisms underlying immune evasion of BCR-ABL1 CML leukemia cells, which sheds light on a combinational treatment for achieving the treatment free remission of CML. Citation Format: Xiaocui Lu, Hui Fang, Xuexiu Fang, Atsuko Matsunaga, Kebin Liu, Jorge Cortes, John K. Cowell, Tianxiang Hu. Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 187.

Abstract 7469: Inflammation plays an important role in the progression of chronic myeloid leukemia

Abstract The development of Tyrosine Kinase Inhibitors (TKIs) resulted in a significant increase in the life expectancy of patients with chronic myeloid leukemia (CML). Nevertheless, TKIs still fail in a significant proportion of therapy-naïve patients, or their effectiveness is hampered by secondary resistance. It is therefore urgent to explore the underlying mechanisms in order to develop new therapeutic approaches to cure CML instead of controlling the disease with a lifelong TKI therapy. Using the ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML we show that BCR::ABL1 leads to the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression. This is supported by our data from CML patients, where we also observe higher levels of pro-inflammatory cytokines in the serum compared to healthy individuals. Furthermore, we demonstrate that BCR::ABL1 drives the expansion of bone marrow derived mast cells (BMMCs) and sensitizes them for FcεRI triggered degranulation. Interestingly, these BMMCs show differences in their lipid metabolism. Hence, sphingomyelin and lysophosphatidylcholine, both known to be involved in inflammation and cancer progression, are upregulated in BCR::ABL1-positive mast cells compared to their negative counterparts. By crossing in the Cpa3Cre mediated model for mast cell deficiency, we demonstrate for the first time that the lack of mast cells prevents the BCR::ABL1 induced upregulation of pro-inflammatory cytokines and also the development of splenomegaly. In addition, we were able to show that splenomegaly in CML patients is associated with high bone marrow mast cell counts and that an upregulation of pro-inflammatory cytokines in patient serum samples correlated with tryptase levels. In summary, our study identifies mast cells and their inflammatory cytokines as important factors in disease progression and suggests considering them as additional targets in CML therapy. Citation Format: Melanie Langhammer, Julia Schöpf, Timo Jaquet, Katharina Horn, Moritz Angel, Daniel Mohl, Simon Lagies, Corinna Spohr, Daniel Christen, Franziska Maria Uhl, Tiago Maié, Henrike Jacobi, Thorsten B. Feyerabend, Julia Huber, Marcus Panning, Cassian Sitaru, Ivan Costa, Robert Zeiser, Konrad Aumann, Heiko Becker, Till Braunschweig, Steffen Koschmieder, Bernd Kammerer, Khalid Shoumariyeh, Michael Huber, Mirle Schemionek-Reinders, Tilman Brummer, Sebastian Halbach. Inflammation plays an important role in the progression of chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7469.

Nilotinib Exerts a Therapeutic Approach via JAK/STAT Pathway and Cytokine Network in Chronic Myeloid Leukemia Cells

Aim: Chronic myeloid leukemia (CML) displays a constitutive tyrosine kinase (TK) activity which in turn leads to the activation of various signaling pathways and the outcome of leukemic phenotype. Activated STAT5A and STAT5B from JAK/STAT pathway induce cell growth, proliferation, differentiation, and survival of leukemic cells which are promoted by a cytokine network. Since the second-generation tyrosine kinase inhibitor nilotinib has the advantage of inhibiting this oncogenic TK activity; we aimed to investigate the underlying mechanism of its therapeutic approach and how it induced apoptosis via analyzing the forthcoming molecular targets of the pathway. 
 Methods: By Nilotinib treatments, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT5A&5B mRNA transcripts, protein expression levels, and also cytokines’ expressional assessments were determined in CML model K562 cells, in vitro.
 Results: Nilotinib treatment in a time and dose-dependent manner assessed a therapeutic approach by decreasing leukemic cell proliferation and survival; inducing leukemic cell apoptosis, down-regulating STAT5A&5B mRNA, and protein expression levels, and regulating cytokine expressional network. 
 Conclusion: Nilotinib-mediated therapeutics could be dependent on targeting JAK/STAT pathway members STAT5A and STAT5B, besides; regulating the cytokine network might be another underlying mechanism for sensitization and response of K562 cells to nilotinib in leukemia pathogenesis.

The molecular signature of BCR::ABLP210 and BCR::ABLT315I in a Drosophila melanogaster chronic myeloid leukemia model

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder resulting from a balanced translocation leading to BCR::ABL1 oncogene with increased tyrosine kinase activity. Despite the advancements in the development of tyrosine kinase inhibitors (TKIs), the T315I gatekeeper point mutation in the BCR::ABL1 gene remains a challenge. We have previously reported in a Drosophila CML model an increased hemocyte count and disruption in sessile hemocyte patterns upon expression of BCR::ABL1p210 and BCR::ABL1T315I in the hemolymph. In this study, we performed RNA sequencing to determine if there is a distinct gene expression that distinguishes BCR::ABL1p210 and BCR::ABL1T315I. We identified six genes that were consistently upregulated in the fly CML model and validated in adult and pediatric CML patients and in a mouse cell line expressing BCR::ABL1T315I. This study provides a comprehensive analysis of gene signatures in BCR::ABL1p210 and BCR::ABL1T315I, laying the groundwork for targeted investigations into the role of these genes in CML pathogenesis.

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

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

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

Acquired microRNA-142 Deficit Drives Escape Mechanisms of Anti-Leukemic Surveillance during Blast Crisis Transformation of Chronic Myeloid Leukemia (CML)

CML may evolve from a chronic phase (CP) into blast crisis (BC), but the underlying mechanisms of the transformation remain to be fully elucidated. We reported that microRNA (miR)-142 is downregulated in BC compared with CP patients; and in a murine model of CP CML (i.e., BCR-ABL mouse), miR-142 knock-out (KO) induced a BC-like phenotype, substantiating a mechanistic role of miR-142 deficit in BC transformation. Mechanistically, miR-142 KO induced mitochondrial fusion and increased oxidative phosphorylation (OxPhos) in leukemic stem cells (LSCs), causing a shift of leukemic phenotype from CP to BC ( Nat Commun, in press). Herein, we report that miR-142 deficit also occurs in T lymphocytes of BC patients due to inflammatory cytokines that are aberrantly produced by the proliferating leukemic cells. Using the Mir142 −/−BCR-ABL mouse, we observed that miR-142 deficit resulted in loss of T cell number and activity, suppressed the antileukemic immune surveillance, and contributed to BC transformation. In fact, miR-142 KO hampered thymic lymphoid-primed multipotent progenitor (LMPP) differentiation into T cells and rendered mature T cells dysfunctional and exhausted, with increase of PD-1 levels, and decrease of the apoptotic threshold, cell cycling and cytokine production, via blockade of OxPhos/glycolysis switch that regulates the metabolism of otherwise activated T cells. These changes translated into a decrease of T-cell antileukemic surveillance as demonstrated by increased numbers of BC murine Lin -Sca-1 +c-Kit + (LSKs) or human CD34+ blasts cocultured with Mir142 −/−T-cells vs those cocultured with Mir142 +/+ T-cells. Furthermore, congenic B6 (lethally irradiated to eradicate host T cells) or immunodeficient NSG (no T cells) recipient mice transplanted with Mir142 −/−BCR-ABL LSK and Mir142 −/− T cells had reduced T cells (both: p<0.0001), increased blasts (B6: p=0.01; NSG: p<0.0001) and a shorter survival (median survival for B6: 58 days vs unreached, p=0.008; for NSG: 28 days vs unreached, p<0.0001) compared with the respective controls transplanted with Mir142 −/−BCR-ABL LSK and Mir142 +/+ T cells. Transplantation of Mir142 −/−BCR-ABL LSKs into Mir142 −/− or Mir142 flox(f)/fLck-cre+ (i.e., miR-142 KO only in T cells; Mir142T Δ/Δ) recipients also resulted in a shorter survival than Mir142 +/+ recipients (median survival for Mir142 −/− vs Mir142 +/+ recipients: 44 vs 53 days, p=0.001; for Mir142T Δ/Δ vs Mir142 +/+ recipients: 42 vs 53 days, p=0.0002). To correct miR-142 deficit, we produced a synthetic miR-142 mimic oligonucleotide (CpG-M-miR-142). We treated a cohort of NSG mice transplanted with Mir142 −/−BCR-ABL LSKs and Mir142 −/− T cells, with CpG-M-miR-142 (30mg/kg/day, IV) or CpG-scramble RNA (SCR) for 3 weeks. We observed increased T cells (p=0.04), decreased blasts (p=0.0029) and longer survival (median survival: unreached vs 37 days, p=0.0005) in CpG-M-miR-142-treated recipients vs SCR-treated controls. Similar results were observed in CpG-M-miR-142-treated Mir142 −/−BCR-ABL mice vs SCR-treated controls. To test CpG-M-miR-142 activity in human T cells, a cohort of BC CML patient-derived xenograft (PDX) mice were given patient's autologous T cells and CpG-M-miR-142. Mice receiving T+CpG-M-miR-142 had increased T cell expansion (p=0.04), reduced leukemic cells (p=0.0078), and longer survival (median survival: 53 vs 38 days, p=0.01) than controls receiving T+SCR. In secondary transplants, the recipients of BM from T+CpG-M-miR-142-treated donors survived longer (median survival: 73 vs 53 days, p<0.0001) than the recipients of BM from T+SCR-treated donors, suggesting a decrease in LSC burden. Finally, we tested the efficacy of tyrosine kinase inhibitors (TKIs), which represent the primary targeting approach for CML, in combination with CpG-M-miR-142 and/or PD-1 inhibitor in Mir142 −/−BCR-ABL mice. The mice treated with TKI+CpG-M-miR-142 (median survival: 107 days, p=0.001) or TKI+PD-1 inhibitor (104 days, p=0.002) survived longer than the controls treated with TKI alone (66 days); the triplet combination (TKI+CpG-M-miR-142+PD-1 inhibitor) is expected to improve survival further (ongoing experiment). In summary, CpG-M-miR-142 alone and in combination with TKI or PD-1 inhibitor rescued T-cell antileukemic activity and prolonged survival in BC CML murine and PDX models, thereby providing a potentially new therapeutic approach for BC CML.

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.

System analysis of Huang-Lian-Jie-Du-Tang and their key active ingredients for overcoming CML resistance by suppression of leukemia stem cells

BackgroundBCR-ABL1-based resistance to imatinib, mainly resulting from BCR-ABL1 mutations, is largely solved after second- and third-generation tyrosine kinase inhibitors (TKIs) are discovered. Nonetheless, imatinib resistance without BCR-ABL1 mutations, including intrinsic resistance induced by stem cells within chronic myeloid leukemia (CML), remains the major clinical challenge for many patients. PurposeTo study the key active ingredients and corresponding target proteins in Huang-Lian-Jie-Du-Tang (HLJDT) against BCR-ABL1-independent CML resistance to therapeutics, and then explore its mechanism of against CML drug resistance. MethodsCytotoxicity of HLJDT and its active ingredients in BCR-ABL1-independent imatinib resistance cells was analyzed through MTT assay. The cloning ability was measured through soft agar assay. Monitoring therapeutic effect on Xenografted mice CML model by in vivo imaging technology and mice survival time. Predicting the potential target protein binding sites by the technology of photocrosslinking sensor chip, molecular space simulation docking, and use Surface Plasmon Resonance (SPR) technology . Flow cytometry to detect the ratio of stem progenitor cells (CD34+). Constructing bone marrow transplantation mice CML leukemia model, detect the effects on leukemia stem cells LSK (Lin-\\ Sca-1+ \\C-kit+) self-renewal. ResultsTreatment with HLJDT, berberine and baicalein inhibited cell viability and colony formation of BCR-ABL1-independent imatinib-resistant cells in vitro while prolonging survival in mouse with CML xenografts and transplatation CML-like mouse models in vivo. JAK2 and MCL1were identified as targets of berberine and baicalein. JAK2 and MCL1 are involved in multi-leukemia stem cell-related pathways. Moreover, the ratio of CD34+ cells in resistant CML cells is higher than in treatment-sensitive CML cells. Treatment with BBR or baicalein partially suppressed CML leukemic stem cells (LSCs) self-renewal in vitro and in vivo. ConclusionFrom the above, we concluded that HLJDT and its key active ingredients (BBR and baicalein) allowed to overcome imatinib resistance with BCR-ABL1 independent by eradication of LSCs by targeting the JAK2 and MCL1 protein levels. Our results lay the foundation for applying HLJDT in patients with TKI-resistant CML.

Mast cell deficiency prevents BCR::ABL1 induced splenomegaly and cytokine elevation in a CML mouse model

The persistence of leukemic stem cells (LSCs) represents a problem in the therapy of chronic myeloid leukemia (CML). Hence, it is of utmost importance to explore the underlying mechanisms to develop new therapeutic approaches to cure CML. Using the genetically engineered ScltTA/TRE-BCR::ABL1 mouse model for chronic phase CML, we previously demonstrated that the loss of the docking protein GAB2 counteracts the infiltration of mast cells (MCs) in the bone marrow (BM) of BCR::ABL1 positive mice. Here, we show for the first time that BCR::ABL1 drives the cytokine independent expansion of BM derived MCs and sensitizes them for FcεRI triggered degranulation. Importantly, we demonstrate that genetic mast cell deficiency conferred by the Cpa3Cre allele prevents BCR::ABL1 induced splenomegaly and impairs the production of pro-inflammatory cytokines. Furthermore, we show in CML patients that splenomegaly is associated with high BM MC counts and that upregulation of pro-inflammatory cytokines in patient serum samples correlates with tryptase levels. Finally, MC-associated transcripts were elevated in human CML BM samples. Thus, our study identifies MCs as essential contributors to disease progression and suggests considering them as an additional target in CML therapy.Mast cells play a key role in the pro-inflammatory tumor microenvironment of the bone marrow. Shown is a cartoon summarizing our results from the mouse model. BCR::ABL1 transformed MCs, as part of the malignant clone, are essential for the elevation of pro-inflammatory cytokines, known to be important in disease initiation and progression.

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

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

Curcumin Decreases Viability and Inhibits Proliferation of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines.

Chronic myeloid leukemia (CML) is a myeloproliferative haematological malignancy characterized by constitutive activation of BCR-ABL1 tyrosine kinase in the majority of patients. BCR-ABL1 expression activates signaling pathways involved in cell proliferation and survival. Current treatment options for CML include tyrosine kinase inhibitors (TKI) with resistance as a major issue. Various treatment options for overcoming resistance are being investigated. Among them, phytochemical curcumin could play an important role. Curcumin has been found to exhibit anti-cancerous effects in various models, including CML, through regulation of multiple molecular signaling pathways contributing to tumorigenesis. We have evaluated curcumin's effects on imatinib-sensitive LAMA84S and K562, as well as imatinib-resistant LAMA84R cell lines. Our results indicate a significant dose-dependent decrease in cell viability and proliferation of imatinib-sensitive and imatinib-resistant cell lines after curcumin treatment. Suppression of key signaling molecules regulating metabolic and proliferative events, such as Akt, P70S6K and NF-kB, was observed. Increased expression of caspase-3 suggests the potential pro-apoptotic effect of curcumin in the imatinib-resistant CML model. Additional in silico molecular docking studies revealed binding modes and affinities of curcumin with different targets and the results are in accordance with in vitro findings. Altogether, these results indicate the potential role of curcumin in the treatment of CML.

A Central Contribution of TG2 Activity to the Antiproliferative and Pro-Apoptotic Effects of Caffeic Acid in K562 Cells of Human Chronic Myeloid Leukemia

Caffeic acid (CA) has shown antitumor activity in numerous solid and blood cancers. We have recently reported that CA is active in reducing proliferation and triggering apoptosis in both Imatinib-sensitive and resistant Chronic Myeloid Leukemia (CML) cells. Tissue transglutaminase type 2 (TG2) enzyme is involved in cell proliferation and apoptosis of numerous types of cancer. However, its activity has different effects depending on the type of tumor. This work investigated the possible involvement of TG2 activation in the triggering of CA-dependent anticancer effects on the K562 cell line, which was studied as a model of CML. CA-dependent changes in TG2 activity were compared with the effects on cell proliferation and apoptosis. The use of N-acetylcysteine (NAC), an antioxidant molecule, suggested that the antiproliferative effect of CA was due to the increase in reactive oxygen species (ROS). The use of a TG2 inhibitor showed that TG2 activity was responsible for the increase in ROS generated by CA and reduced both caspase activation and triggering of CA-dependent apoptosis. The knocking-down of TGM2 transcripts confirmed the crucial involvement of TG2 activation in CML cell death. In conclusion, the data reported, in addition to ascertaining the important role of TG2 activation in the antiproliferative and pro-apoptotic mechanism of CA allowed us to hypothesize a possible therapeutic utility of the molecules capable of triggering the activation pathways of TG2 in the treatment of CML.

Targeting HIF-2α for the Treatment of CML By Affecting LSCs Metabolism and the Vascular Microenvironment

Although the advent of tyrosine kinase inhibitors (TKI) drugs significantly increased the long-term survival of chronic myeloid leukemia (CML), residual LSCs can still lead to drug resistance and disease recurrence. As LSCs in CML rely on hypoxia-inducible factors (HIF) to maintain their own energy metabolism in the hypoxic environment of bone marrow, HIF-2α has not been systematically explored in CML. Here, we explored how HIF-2α participated in drug resistance and provided a potential new clinical strategy for CML. We first investigated the expression of HIF in the database and found the overexpression of HIF-2α in the bone marrow of CML patients compared with normal subjects, while there was no difference in HIF-1α and HIF-3α (Fig. 1A-B). We then collected bone marrow samples from normal subjects and CML patients. We found that compared with normal hematopoietic stem cells (Lin-CD90+CD34+CD38-), CML LSCs (Lin-CD90+CD34+CD38-) had higher levels of HIF-related proteins than common leukemia cells (Fig. 1C). To further explore the role of HIF-2α in the pathogenesis of CML, we constructed HIF-2α conditional knockout mice through transfecting the plasmid of Cre-BCR-ABL-GFP with transposase and Ctrl-BCR-ABL-GFP without transposition to HSPCs (Fig. 2A). After the first round of leukemia transplantation, the onset of CML was delayed in the HIF-2α knockout mice and the median survival time was prolonged from 31d in the Ctrl group to 60d (Fig. 2B). Next, we harvested fully onset CML mice for the secondary transplantation and found the median survival time extended from 46d to 117d in the HIF-2α knockout group (Fig. 2C). Then we investigate the biological function of HIF-2α in CML LSCs. We performed the clone formation assay on bone marrow cells of the first round of onset CML mice and found that HIF-2α knockout reduced the colony-forming ability of CML cells (Fig. 3A). To explore the tumorigenic ability of HIF-2α knockout, limiting dilution assay (LDA) was performed and found that the LSCs ratio of CML decreased by 30 times after HIF-2α knockout, from 1/76960 to 1/2237970 (Figure. 3B). Meanwhile, we explored the effect of HIF-2α knockout on the chemosensitivity in CML. TKI treatment combined with HIF-2α knockout can significantly improve the efficacy of TKI and extend the mouse survival time (Figure. 3C). To explore the specific mechanism by which HIF2A affects LSCs, we firstly investigate the effect of HIF-2α on LSCs metabolism. We detected metabolism-related indexes of LSCs in the CML model and found that the glycolysis ability of LSCs was weakened after HIF-2α knockout (Fig. 4A). At the same time, its oxidative phosphorylation pathway and reactive oxygen species (ROS) were enhanced (Fig. 4B-C) which may be an important cause of LSCs apoptosis. As HIF-2α can directly promote the transcription of pro-angiogenic factors, we detected the content of vascular endothelial growth factor (VEGF) α in the supernatant of mice bone marrow and found that it was significantly increased after the onset of CML but decreased after HIF-2α knockout (Fig. 5A). Fluorescent staining of bone marrow sections revealed that the density of blood vessels in bone marrow increased, whether in the intima (Fig. 5B) after the onset of CML. Next, we treated CML mice with VEGF Monoclonal antibody and found that it alone could prolong the survival time. However, the VEGF Monoclonal antibody did not affect HIF-2α knockout CML mice (Fig. 5C). These results suggest that HIF-2α is involved in bone marrow vascular remodelling after the onset of CML. To investigate the functional effect of HIF-2α in human CML, the CDX CML mouse model was made with CML cell lines (K562 and MEG-01) to observe the tumorigenesis of CML in nude mice, and they were treated with TKI. We found that HIF-2α knockout could delay the onset of CML and improve the therapeutic effect of TKI (Fig. 6A). To better apply the effect of HIF-2α on CML chemosensitivity to the clinic, we explored the effect of inhibitors of HIF, PT2399, on CML chemosensitivity. PT2399 can significantly increase the chemosensitivity of CML cell lines to TKIs and prolong the survival curve of CML mice treated with TKIs (Fig. 6B). In conclusion, HIF-2α knockout could inhibit the function of LSCs by affecting their metabolic pattern and vascular microenvironment in CML. Therefore, targeting HIF-2α may become a new strategy, and its specific inhibitor PT2399 may be a candidate drug for clinical application in CML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Elvn-001, a Next Generation ATP-Competitive ABL1 Tyrosine Kinase Inhibitor for the Treatment of Chronic Myeloid Leukemia

Of the six small-molecule ABL1 tyrosine kinase inhibitors (TKIs) that have received FDA-approval for the treatment of BCR-ABL1-driven Chronic Myeloid Leukemia (CML), five (Imatinib, Nilotinib, Dasatinib, Ponatinib, Bosutinib) target the ATP-binding site in the kinase domain. While each of these TKIs provides added value to the arsenal of treatments for CML, these agents suffer from selectivity issues, inhibiting a number of off-target kinases that drive an array of dose-limiting toxicities (DLTs) which constrain their ability to fully engage ABL1 and drive maximal efficacy. In addition, the emergence of on-target resistance against these TKIs, such as the highly prevalent T315I mutation at the gatekeeper position of the active site, is common and represents a significant clinical challenge. The recently approved ABL1 TKI Asciminib is an allosteric inhibitor that interacts with the unique myristoyl binding pocket of ABL1 and therefore possesses high kinome selectivity and an improved tolerability profile. Despite its unique binding mode, clinical activity against the T315I mutant requires a dose five times higher than that approved for the broader third-line or greater patient population, which leads to issues with tolerance. In addition, emergent on-target resistance remains a challenge, particularly with respect to mutations within the myristoyl pocket as well as select active site mutants. Here we report the discovery of ELVN-001, a potent ATP-competitive small-molecule ABL1 TKI which, unlike the majority of currently approved ABL1 TKIs, is highly selective in both biochemical and cell-based assays versus the kinome in general as well as the key off-target kinases KDR, c-KIT, PDGFR and SRC, which are associated with a variety of DLTs including myelosuppression, edema, pleural effusions, hypertension, and certain cardiovascular events. ELVN-001 exhibited potent pharmacodynamic (phosphorylated CRKL) and anti-proliferative activity in vitro against BCR-ABL1-driven CML cell lines with comparable or superior activity to the approved therapies. Evaluation of ELVN-001 against Ba/F3 cell lines expressing a panel of clinically relevant BCR-ABL1 TKI-resistant mutants demonstrated activity of ELVN-001 against many of these mutants, including T315I as well as two myristoyl pocket mutants (A334P, P465S) which are highly resistant to asciminib. Cell-based mutagenesis screens using Ba/F3 BCR-ABL1 cells confirmed a concentration-dependent reduction in outgrowth of resistant clones, which generally narrowed to a select subset of P-loop mutations. Consistent with previously reported findings for ponatinib, while ELVN-001 showed little to no activity against cells expressing a subset of T315I-inclusive BCR-ABL1 compound mutants, the addition of Asciminib resulted in varying degrees of re-sensitizing these mutants to ELVN-001. Importantly, ELVN-001 possessed a favorable absorption, distribution, metabolism, elimination, and toxicity profile, as determined from both in vitro and in vivo studies, that readily translated into favorable pharmacokinetic properties in all preclinical species evaluated. Accordingly, administration of doses of ELVN-001 corresponding to well-tolerated free drug exposures in rats and non-human primates readily translated to mouse in vivo tumor xenograft models of efficacy, including a model of T315I CML. These anti-tumor activities also compared favorably to currently approved therapies when evaluated head-to-head in these studies. Together, these findings support ELVN-001 as an extremely selective, novel ATP-competitive inhibitor of ABL1 kinase with superior pharmacokinetic and tolerability properties compared to currently approved TKIs and which retains activity against T315I and other key on-target resistance mutations, warranting its further investigation in patients with TKI-resistant/intolerant CML.

Liver Toxicity As Dose Limiting Toxicity of Bosutinib in Combination with Atezolizumab in Chronic Myeloid Leukemia. Results from the Phase Ib/II Zerolmc Trial

INTRODUCTION: Despite the current excellent prognosis of chronic myeloid leukemia (CML) patients, a significant percentage of patients will not achieve a deep molecular response that would allow them to discontinue treatment. Immune cell-mediated approaches such as allogeneic hematopoietic cell transplantation and interferon were the mainstays for the treatment of CML before tyrosine kinases inhibitors arrival. The programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis has been shown as a potential target in CML in murine model of CML. A previous study showed an adequate safety profile of dasatinib in combination with nivolumab in CML pts failing to previous TKIs. AIMS: To evaluate the safety and efficacy of bosutinib in combination with atezolizumab for CML newly diagnosed patients. PATIENTS AND METHODS: Phase Ib/II, single arm, open-label and dose-escalation study designed to determine the safety profile and the recommended phase III dose (RP3D) of bosutinib when administered in combination with atezolizumab in naïve patients with chronic myeloid leukemia in chronic phase (Phase Ib/II). The primary endpoint was to determine the safety and tolerability of the combination therapy. Secondary endpoints were the number and rate of patients that achieve major molecular response MMR. Treatment schedule was designed in 3 phases. First therapy: 400 mg of bosutinib/day for one treatment cycle (28 days). Second therapy: 400 mg of bosutinib/day combined with either 840 mg/2 weeks or 1680 mg/4 weeks of atezolizumab for 12 cycles. Third therapy: 400 mg of bosutinib/day for 12 cycles. The incidence, nature, and severity of adverse events (AEs), serious AEs (SAEs), and AEs of special interest (AESIs) were graded according to the National Cancer Institute Common Terminology Criteria for AE (version 4). If dose-limiting toxicities (DLTs) appeared in 2 or more patients the study was terminated. RESULTS: A total of 36 patients were initially planned to be enrolled. Due to premature study discontinuation, only 9 patients were finally recruited. 8 of them reached the 2nd therapy phase of the study and received, at least one dose of atezolizumab in combination with bosutinib. 3 patients receive one dose of 840 mg of atezolizumab, 3 patients receive 2 doses of 840 mg of atezolizumab, and 2 patients receive 3 doses of 840 mg of atezolizumab. The onset of several events affecting the liver normal function (elevation of transaminases), considered AESIs, led to the premature termination of the study after the onset 2 DLTs. Totally, 4 (50% of the patients that received treatment combination) patients withdrew due to liver toxicity (grade 3-4) (table 1). No patients suffered from liver comorbidities nor autoimmune disorders previous to study entry. One patient suffered from liver toxicity in the bosutinib monotherapy phase. All patients completely recovered after treatment interruption. Altogether, 55 AEs were reported during the study (table 1), 35 (63.6% of the total) were mild or grade 1, 5 (9.1%) moderate or grade 2, 12 (21.8%) were severe or grade 3 and 3 (5.5%) were considered life-threatening, grade 4 events, Alanine aminotransferase increased, Cholestasis and Blood bilirubin increased. The AEs were mainly gastrointestinal disorders (N=26), specially, diarrhoea (N=11), vomiting (N=6) and nausea (N=4). Of those, possibly related AEs, 19 were mild, 4 moderate, 10 severe and 3 life threatening, all 3 detected in the same patient. Regarding efficacy, at cycle 4 (3 months follow up), 100% of the patients tested (n=3) reached complete cytogenetic response. CONCLUSIONS: We found dose limiting toxicity when combining bosutinib with atezolizumab due to serious liver toxicities events. The use of immune check points inhibitors could increase adverse events related to TKIs. This information should be taken into consideration when designing clinical trials combining these two treatments options. This study is sponsored by Pfizer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 15594: Decreasing the High Risk of Ponatinib-Induced Cardiovascular Toxicity in Chronic Myeloid Leukemia

Introduction: Cardiovascular (CV) toxicity of cancer therapeutics is a growing problem. Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs) and is used as a highly effective TKI against ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases CV events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. There is need of novel therapeutics to provide treatment for CML while avoiding CV toxicity. Hypothesis: Using in vitro and in vivo models of CV toxicity can make possible to reengineer ponatinib to minimize its toxicity. Methods: We developed new safer analogues of ponatinib using fragment-based approach. Their anti-tumor efficacy was tested in 2 different CML cell lines and CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs), and for bone marrow toxicity by colony formation assay in human bone marrow cells (HBMCs). Finally, we confirmed the safer CV profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs, vasculogenesis in HMVECs and bone marrow toxicity in HBMCs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, CV-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal CV toxicity compared to ponatinib.

INFLAMMATORY CYTOKINES PERTURB NK CELL FUNCTION AND GENE SIGNATURE IN CHRONIC MYELOID LEUKEMIA

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent anti-cancer immunotherapeutic. NK cells'mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in CML patients. The molecular drivers of this impairment during leukemic progression remain unclear. Given the established role of inflammatory cytokines (namely IL1β, IL-6, GM-CSF, TNFα) in driving myeloid malignancies, we aimed to define their effect on NK cell phenotype and function using a unique pre-clinical chimeric mouse model of CML. We hypothesize that leukemic cytokines impede NK cell maturation and cytotoxicity and shift NK cells toward a pro-tumor cytokine-producing phenotype. We establish a chimeric mouse model of CML by transplanting bone marrow BCR-ABL+ CD45.2 cells into sub-lethally irradiated CD45.1 C57BL/6 wild-type mice. For controls, mice are transplanted with non-mutant cells. Such chimeras represent a robust model to study the exposure of non-transformed host cells to leukemia. At the endpoint, splenic, blood, and bone marrow NK cells are harvested and profiled by flow cytometry, RT-qPCR, scRNA-seq, or used for degranulation assay. Blood samples are collected to obtain serum. Consistent with clinical observations, NK cells are severely reduced in numbers and frequencies and display an immature phenotype and diminished expression of activating receptors (Nkp46, Ly49D) while upregulating inhibitory molecules (Lag-3, TIGIT, NKG2A) during leukemia. Moreover, CML-exposed NK cells have a higher proliferation rate and a greater cell death indicating perturbed homeostasis. We revealed a preferential loss of terminally mature CD27−CD11b+ NK subset in CML mice, suggesting a functional shift from cytotoxic phenotype toward the immature cytokine-producing. In agreement, CML-exposed NK cells show impaired cytotoxicity as measured by target-specific degranulation ex vivo . Next, we mapped the transcriptional landscape of CML-exposed NK cells using single-cell RNA-sequencing. We confirmed decreased gene expression of NK maturation and cytotoxicity markers ( Itgam, Cx3cr1, Prf1, Gzma ) and upregulation of inhibitory molecules ( Tigit, Lag-3, Pdcd1lg1/2) and proteins involved in the cell cycle and senescence ( Mki67, Ccna, Ccnb ). Importantly, CML-exposed NK cells had significantly increased mRNA levels of inflammatory cytokines including IL1β, TNF, GM-CSF; cytokine receptors Il6st, Il-2Ra; and negative regulators of STAT signaling SOCS1/2/3 and CIS. Furthermore, IL-6-STAT3 and IL1β/TNF-signaling gene signatures were enriched in CML-exposed NK cells – an effect that is likely to be triggered by inflammatory cytokines. Thus, we next found that peripheral blood serum from CML mice dampens healthy NK cell degranulation ex vivo , compared to serum from control mice. Further ELISA analysis revealed elevated levels of IL-6, IL-1, TNF, and GM-CSF in CML serum samples. Some of these cytokines might be secreted by functionally skewed NK cells, as suggested by gene expression analysis. Altogether, our data suggest that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing optimal targets for NK-boosting CML immunotherapies.

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

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

Abstract P3105: Improving Cardiovascular Toxicity Of Chronic Myeloid Leukemia Therapy.

Introduction: Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects. Hypothesis: Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity. Methods: Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.