Chronic Myeloid Leukemia Patient Samples Research Articles

Abstract 4404: EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2

Abstract The polycomb protein EZH2 is a commonly deregulated or mutated transcriptional regulator in various cancers. In Chronic Myeloid Leukemia (CML) it has been reported to be upregulated and associated with transcriptional reprograming, rendering CML leukemic stem cells (LSCs) sensitive to EZH2 inhibitor treatment. Mechanistically this seems to be linked to the silencing of apoptotic target genes by EZH2-mediated histone H3 lysine 27 tri-methylation (H3K27me3). We hypothesized that the regulation of apoptotic genes is not the only pathway responsible for its oncogenic function, and that EZH2 might also act as a modulator of the mRNA splicing landscape. To test this, we treated the CML cell line K562 with the EZH2 inhibitors GSK126 and EPZ-6438 and performed RNA-seq followed by splicing analysis. Using two different bioinformatics tools, we detected differential splicing of several hundreds of splicing events. However, differentially spliced genes were not associated with increased EZH2 or H3K27me3 signals, suggesting an indirect mechanism of splicing regulation. Indeed, we found 11 splicing factors transcriptionally upregulated upon EZH2 inhibitor treatment. CELF2 was identified as top candidate to mediate part of the EZH2 inhibitor induced phenotype. Upon overexpression, we observed 1) reduced cell growth, viability, and colony formation of K562 cells, 2) a change in the splicing landscape, significantly overlapping with EZH2 inhibitor induced changes, 3) the downregulation of cMYC signaling, also found upon EZH2 inhibitor treatment. To test if CELF2 is indeed acting as EZH2-regulated tumor suppressor in CML, we extended our study to CML patient samples: we performed RNA-seq followed by splicing and gene expression analysis in seven CML patient-derived CD34+ samples and eight healthy donor-derived CD34+ samples, as well as three CML patient samples transduced with CELF2-containing expression constructs. While we found Celf2 mRNA downregulated in CML derived samples, overexpression led to decreased colony formation and cMYC signaling, validating our findings from K562 cells. We also performed an analysis of CML associated splicing changes and found hundreds of deregulated splicing events, of which a significant portion overlapped with CELF2 overexpression mediated changes, suggesting direct regulation by CELF2. Based on this data we propose a novel role of EZH2, where it exerts part of its oncogenic function in CML through the transcriptional repression of splicing factors, in particular CELF2, leading to splicing deregulation, increased cMYC signaling, and enhanced cell growth and viability. Finally, data from other publicly available EZH2 inhibitor treated cancer models suggests that splicing modulation by EZH2 might not be restricted to CML. Citation Format: Reinhard Brunmeir, Ying Li, Baohong Lin, Leong Qiao Zheng, Henry Yang, Wee Joo Chng. EZH2 modulates mRNA splicing in chronic myeloid leukemia through the repression of splicing factors and exerts part of its oncogenic function through the regulation of CELF2 [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 4404.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.

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.

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.

The Molecular Role of HIF1α Is Elucidated in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is potentially fatal blood cancer, but there is an unmet need to discover novel molecular biomarkers. The hypothesis of this study aimed to elucidate the relationship of HIF1α with the redox system, Krebs cycles, notch1, and other regulatory proteins to better understand the pathophysiology and clinical relevance in chronic myeloid leukemia (CML) patients, as the molecular mechanism of this axis is still not clear. This study included CML patient samples (n = 60; 60: blood; 10: bone marrow tissues) and compared them with healthy controls (n = 20; blood). Clinical diagnosis confirmed on bone marrow aspiration, marrow trephine biopsy, and BCR/ABL1 translocation. Cases were subclassified into chronic, accelerated, and blast crises as per WHO guidelines. Molecular experiments included redox parameters, DNA fragmentation, Krebs cycle metabolites, and gene expression by RT-PCR/Western blot/LC-MS, PPI (STRING), Pearson correlation, and ROC curve analysis. Here, our findings show that p210/p190BCR/ABL1 translocation is common in all blast crisis phases of CML. Redox factor/Krebs oncometabolite concentrations were high, leading to upregulation and stabilization of HIF1α. HIF1α leads to the pathogenesis in CML cells by upregulating their downstream genes (Notch 2/4/Ikaros/SIRT1/Foxo-3a/p53, etc.). Whereas, downregulated ubiquitin proteasomal and apoptotic factors in CML pateints, can trigger degradation of HIF1α through proline hydroxylation. However, HIF1α showed a negative corelation with the notch1 pathway. Notch1 plays a tumor-suppressive role in CML and might have the potential to be used as a diagnostic marker along with other factors in CML patients. The outcome also revealed that oxidant treatment could not be effective in augmentation with conventional therapy because CML cells can enhance the levels of antioxidants for their survival. HIF1α might be a novel therapeutic target other than BCR/ABL1 translocation.

Restoration of miR-23a expression by chidamide sensitizes CML cells to imatinib treatment with concomitant downregulation of CRYAB

ABSTRACT MicroRNAs (miRNAs) are involved in various processes from the initiation and development of cancers, including chronic myeloid leukemia (CML). In this report, we aimed to investigate the roles of miR-23a in the regulation of imatinib mesylate (IM) sensitivity in CML cells and the possible mechanisms involved in this process. We demonstrated that the expression of miR-23a was markedly low in bone marrow mononuclear cells from patients in whom IM treatment had failed and imatinib-resistant K562/G01 cells when compared to patients with optimal responses and imatinib-sensitive K562 cells, respectively. Overexpression of miR-23a was shown to induce apoptosis of K562/G01 cells and sensitize these cells to imatinib treatment. With the aid of bioinformatics analysis, we revealed that CRYAB could be a potential downstream effector of miR-23a, contributing to miR-23a-mediated IM resistance. We also observed that the expression of CRYAB was inversely correlated with miR-23a expression in CML cell lines and patient samples. Importantly, chidamide upregulated miR-23a expression and reversed the IM resistance of CML cells. Together, these findings strongly suggest that miR-23a acts as a tumor suppressor by downregulating CRYAB expression. Restoration of miR-23a by chidamide may therefore have a therapeutic effect in controlling the sensitivity of CML cells to imatinib.

A Role for Lipid Metabolism in Tyrosine Kinase Inhibitor (TKI) Resistance of Chronic Myeloid Leukemia (CML)

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p<0.02) (Gonzalez MA, et al. Cancer Res 2020 #648). Low G0S2 expression not only correlated with TKI resistance, but also with a shorter overall survival in chronic phase (p=0.036), and transformation from the chronic to the blast phase of disease (p<0.05). G0S2 is known to regulate apoptosis, quiescence, lipid metabolism, oxidative phosphorylation, and also acts as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in intracellular lipolysis. We hypothesized that G0S2-mediated ATGL inhibition underlies its tumor suppressor activity in CML and TKI resistance.To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p<0.0001). These data suggest that ATGL abrogates G0S2-mediated apoptosis in the presence of imatinib. We next performed colony formation assays of K562 cells with G0S2 knockdown (shG0S2) versus a non-targeting control (shNT) in the absence and presence of an ATGL inhibitor (Atglistatin, 50 µM) and a fatty acid oxidation (FAO) inhibitor (TMZ, 10 µM). Atglistatin had no effect on colony formation in the shNT-expressing cells, but reduced survival by ~40% upon G0S2 knockdown. Similarly, TMZ reduced survival of shNT cells by ~30%, while it reduced survival by ~90% upon G0S2 knockdown. These data suggest that the phenotypic effects of G0S2 knockdown in TKI resistance in part requires FAO.The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. DisclosuresNo relevant conflicts of interest to declare.

Plasma-Derived 3D Culture for Leukemic Proliferation

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

Discovery of a Novel CIP2A Variant (NOCIVA) with Clinical Relevance in Predicting TKI Resistance in Myeloid Leukemias.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that inhibits the tumor suppressor PP2A-B56α. However, CIP2A mRNA variants remain uncharacterized. Here, we report the discovery of a CIP2A splicing variant, novel CIP2A variant (NOCIVA). Characterization of CIP2A variants was performed by both 3' and 5' rapid amplification of cDNA ends from cancer cells. The function of NOCIVA was assessed by structural and molecular biology approaches. Its clinical relevance was studied in an acute myeloid leukemia (AML) patient cohort and two independent chronic myeloid leukemia (CML) cohorts. NOCIVA contains CIP2A exons 1 to 13 fused to 349 nucleotides from CIP2A intron 13. Intriguingly, the first 39 nucleotides of the NOCIVA-specific sequence are in the coding frame with exon 13 of CIP2A and code for a 13-amino acid peptide tail nonhomologous to any known human protein sequence. Therefore, NOCIVA translates to a unique human protein. NOCIVA retains the capacity to bind to B56α, but, whereas CIP2A is predominantly a cytoplasmic protein, NOCIVA translocates to the nucleus. Indicative of prevalent alternative splicing from CIP2A to NOCIVA in myeloid malignancies, AML and CML patient samples overexpress NOCIVA, but not CIP2A mRNA. In AML, a high NOCIVA/CIP2A mRNA expression ratio is a marker for adverse overall survival. In CML, high NOCIVA expression is associated with inferior event-free survival among imatinib-treated patients, but not among patients treated with dasatinib or nilotinib. We discovered a novel variant of the oncoprotein CIP2A and its clinical relevance in predicting tyrosine kinase inhibitor therapy resistance in myeloid leukemias.

TIF-Seq2 disentangles overlapping isoforms in complex human transcriptomes.

Eukaryotic transcriptomes are complex, involving thousands of overlapping transcripts. The interleaved nature of the transcriptomes limits our ability to identify regulatory regions, and in some cases can lead to misinterpretation of gene expression. To improve the understanding of the overlapping transcriptomes, we have developed an optimized method, TIF-Seq2, able to sequence simultaneously the 5′ and 3′ ends of individual RNA molecules at single-nucleotide resolution. We investigated the transcriptome of a well characterized human cell line (K562) and identified thousands of unannotated transcript isoforms. By focusing on transcripts which are challenging to be investigated with RNA-Seq, we accurately defined boundaries of lowly expressed unannotated and read-through transcripts putatively encoding fusion genes. We validated our results by targeted long-read sequencing and standard RNA-Seq for chronic myeloid leukaemia patient samples. Taking the advantage of TIF-Seq2, we explored transcription regulation among overlapping units and investigated their crosstalk. We show that most overlapping upstream transcripts use poly(A) sites within the first 2 kb of the downstream transcription units. Our work shows that, by paring the 5′ and 3′ end of each RNA, TIF-Seq2 can improve the annotation of complex genomes, facilitate accurate assignment of promoters to genes and easily identify transcriptionally fused genes.

Abstract 648: Role of G0S2 in chronic myeloid leukemia and TKI resistance

Abstract Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. However, resistance is a clinical problem, and TKIs do not target CML leukemic stem cells (LSC), which are independent from BCR-ABL1 kinase activity. To understand mechanisms driving BCR-ABL1-independent resistance, we analyzed the transcriptional signature of TKI-naïve CD34+ cells from patients with or without a response to imatinib after 12 months of therapy (McWeeney et al. Blood 2010). Among the genes most profoundly downregulated in patients destined to fail imatinib was G0/G1 switch gene 2 (G0S2) (&amp;gt;3-fold, n=59, p&amp;lt;0.02). Retrospective analysis of survival data for patients whose samples were evaluated in a microarray revealed that high G0S2 expression (&amp;gt;50%) correlated with a longer overall survival (n=30 responders, n=16 non-responders, p=0.036). We next analyzed G0S2 mRNA expression in CD34+ cells from normal cord blood and from primary CD34+ cells lacking BCR-ABL1 kinase domain mutations. G0S2 was 4-fold downregulated in newly diagnosed CML (n=6) compared to normal cord blood (n=5, p&amp;lt;0.01), and further downregulated by &amp;gt;3-fold in TKI-resistant (n=2) and BP-CML samples (n=5, p&amp;lt;0.01).G0S2 mRNA was also lower in CD34+38- stem cells compared to committed CD34+38+ progenitor cells in CML (n=6, p&amp;lt;0.01) but not normal cord blood (n=4, p=ns). CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions. To assess the role of G0S2 in CML, we cloned G0S2 amplified from normal human mononuclear cells into a lentiviral expression system, and confirmed ectopic expression by qRT-PCR and immunoblot. Ectopic G0S2 reduced colony formation in both TKI-sensitive and TKI-resistant CML cell lines and CD34+ CML patient samples. While G0S2 has been reported to induce apoptosis by direct inhibition of BCL-2, reduced colony formation was not associated with an increase of apoptosis, but did restore imatinib-induced apoptosis. These data are consistent with a tumor suppressor role for G0S2 in CML. G0S2 has also been shown to inhibit adipocyte triglyceride lipase (ATGL), the rate-limiting enzyme in the conversion of triglycerides to free fatty acids (FFAs). While ATGL is indeed expressed at the protein level in CML cells, shRNAs targeting ATGL do not phenocopy ectopic G0S2 expression. Furthermore, the effects of ectopic G0S2 are not impaired in cells with simultaneous ATGL knockdown, and ectopic G0S2 has no effect on intracellular FFA or ATP levels. Finally, G0S2 gene expression has been shown to be regulated by promoter DNA hypermethylation. However, DNA bisulfite and patch PCR sequencing in primary CD34+ cells from CML patients did not reveal G0S2 promoter hypermethylation in CML. Rather, ChIP-PCR revealed the presence of MYC/MAX at the G0S2 promoter in CML, suggesting MYC-mediated transcriptional repression. Altogether, these data suggest that G0S2 downregulation plays a functional role in CML disease progression and TKI response, but is independent from its role as an inhibitor of BCL-2 or ATGL. These data unravel a new role for G0S2 as a regulator of TKI response in CML, and suggest that restoring G0S2 expression may have clinical utility. Citation Format: Mayra A. Gonzalez, Alfonso E. Bencomo, Christian Barreto-Vargas, Andres J. Rubio, Idaly M. Olivas, Joshua J. Lara, Anna Senina, Jonathan Ahmann, Katherine T. Varley, Luis F. Jave-Suarez, O'Hare Thomas, Michael W. Deininger, Anna M. Eiring. Role of G0S2 in chronic myeloid leukemia and TKI resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 648.

CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy

The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed “minimal residual disease”. The phenomenon of disease persistence suggests that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of leukemic stem cells (LSCs). Although other markers of a primitive CML LSC population have been identified in the preclinical setting, only CD26 appears to offer clinical utility. Here we demonstrate consistent and selective expression of CD93 on a lin−CD34+CD38−CD90+ CML LSC population and show in vitro and in vivo data to suggest increased stem cell characteristics, as well as robust engraftment in patient-derived xenograft models in comparison with a CD93− CML stem/progenitor cell population, which fails to engraft. Through bulk and single-cell analyses of selected stem cell and cell survival-specific genes, we confirmed the quiescent character and demonstrate their persistence in a population of CML patient samples who demonstrate molecular relapse on TKI withdrawal. Taken together, our results identify that CD93 is consistently and selectively expressed on a lin−CD34+CD38−CD90+ CML LSC population with stem cell characteristics and may be an important indicator in determining poor TKI responders.

Inhibition of KPNB1 Inhibits Proliferation and Promotes Apoptosis of Chronic Myeloid Leukemia Cells Through Regulation of E2F1.

BackgroundKaryopherin-β1 (KPNB1) belongs to the karyopherin superfamily, which functions as shuttling proteins from the cytoplasm to nuclear. A high level of KPNB1 has been reported in various cancers which promotes cell proliferation and inhibits apoptosis. However, the role of KPNB1 in chronic myeloid leukemia (CML) remains uncertain.MethodsExpression level of KPNB1 in CML patient samples and cell lines was analyzed by Western blotting. The proliferation assays and colony formation assay were used to study the CML cell proliferation when KPNB1 knockdown in vitro. Next, Western blotting was used to evaluate the effects of KPNB1 on E2F1 and other cell cycle regulators. Then, the location of E2F1 was detected by immunofluorescence. Finally, flow cytometry was used to detect the effect of KPNB1 inhibitor importazole (IPZ) on CML cells.ResultsIn this study, we firstly showed that KPNB1 is over-expressed in CML cells. Targeting KPNB1 with small interfering RNA (siRNA) and IPZ reduced proliferation and induced apoptosis of CML cells. The underlying mechanisms were also investigated that E2F1 nuclear transport was blocked after inhibiting KPNB1 with siRNA, suggesting KPNB1 over-expression mediates the excessive nuclear transport of E2F1 in CML cells. Moreover, the expression of the E2F1 targeted molecule such as c-Myc and KPNA2 was markedly reduced. The IPZ arrested CML cells at G2/M phase and induced cell apoptosis.ConclusionIn summary, our results clearly showed that KPNB1 is over-expressed in CML cells and mediates the translocation of E2F1 into the nucleus of CML cells, thereby inhibition of KPNB1 reduced proliferation and induced apoptosis of CML cells which provides new insights for targeted CML therapies.

Histone chaperone HIRA dictate proliferation vs differentiation of chronic myeloid leukemia cells.

Abnormal proliferation and disrupted differentiation of hematopoietic progenitors mark leukemia. Histone cell cycle regulator A (HIRA), a histone chaperone, regulates hemogenic to hematopoietic transition involved in normal hematopoiesis. But, its role remains unexplored in leukemia, a case of dysregulated hematopoiesis. Here, the Cancer Cell Line Encyclopedia database analysis showed enhanced HIRA mRNA expression in cells of hematopoietic and lymphoid origin with maximal expression in the chronic myeloid leukemia (CML) cell line, K562. This observation was further endorsed by the induced expression of HIRA in CML patient samples compared to healthy individuals and Acute Myeloid Leukemia patients. Downregulation of HIRA in K562 cells displayed cell cycle arrest, loss in proliferation, presence of polyploidy with significant increase in CD41+ population thereby limiting proliferation but inducing differentiation of leukemia cells to megakaryocyte fate. Induced megakaryocyte differentiation of mouse Hira‐knockout hematopoietic progenitors in vivo further confirmed the in vitro findings in leukemia cells. Molecular analysis showed the involvement of MKL1/GATA2/H3.3 axis in dictating differentiation of CML cells to megakaryocytes. Thus, HIRA could be exploited for differentiation induction therapy in CML and in chronic pathological conditions involving low platelet counts.

S127 CHRONIC MYELOID LEUKEMIA IS ASSOCIATED WITH INCREASED NEUTROPHIL EXTRACELLULAR TRAP FORMATION THAT IS AUGMENTED BY PONATINIB – A POTENTIAL CONTRIBUTOR TO VASCULAR TOXICITY?

Background:Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm (MPN) driven by the occurrence of the bcr‐abl1 fusion gene. Cardiovascular (CVS) and thrombotic complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKI's) used to treat CML such as ponatinib. The mechanism underlying these CVS adversities is not fully understood but may involve off‐target effects that alter the function of vascular endothelial cells as well as other cells involved in the pathogenesis of thrombosis.More than a decade ago Brinkmann and collegues (Science, 2004) described the occurance of neutrophil extracellular traps (NETs). In response to various stimulli, neutrophils can expell extracellular strands of decondensed DNA in complex with histones and other neutrophil granular proteins. These structures have the ability to ensnare and kill microbes but are also implicated in the pathogenesis of autoimmunity and thrombosis. Previous studies demonstrated that NETs have a role in the increased thrombotic tendency associated with Philadelphia‐negative MPNs (Wolach et al, STM 2018).Aims:To study NET formation in CML and evaluate the possible effect of the different TKI's on NET formation.Methods:Neutrophils were isolated from patients with CML (n = 7) and from age and gender matched controls (n = 7) by Ficoll gradient. NET formation was analyzed in‐vitro following neutrophil stimulation with phorbol 12‐myristate 13‐acetate (PMA), ionomycin and lipopolysaccharides (LPS) and assessed by the NETosis Assay Kit (Cayman chemical) as well as by immunofluorescence (IF) studies. PAD4 and citrullinated H3 (H3cit) expression was determined by Western blot. Membrane expression of CD11b and reactive oxygen species (ROS) production were analyzed by flow cytometry. NET formation and ROS production were assessed in resting and stimulated conditions and after ex‐vivo pre‐treatment of neutrophils with clinically relevant concentrations of various TKI's.Results:Neutrophils from patients with CML had increased NET formation at baseline (p = 0.03) and after stimulation with PMA (p = 0.002). Pretreatment of neutrophils with ponatinib significantly augmented PMA‐induced NET formation as compared to DMSO, imatinib and dasatinib treated neutrophils (p = 0.05 for all comparisons). ROS generation was significantly enhanced in CML patient samples compared to controls (p &lt; 0.01). Ponatinib treatment also led to an increase of ROS levels in neutrophils from patients with CML. PAD4 and H3cit expression was higher in neutrophil lysates form patients with CML (n = 3) as compared to controls (n = 3), (p &lt; 0.05, p &lt; 0.01, respectively).Summary/Conclusion:CML is associated with an increase in NET formation which is further augmented by ponatinib. Further studies are needed to assess the possible role of increased NET formation in CML on the CVS and thrombotic complications associated with ponatinib exposure.image

PF466 POTENTIAL COMBINATIONAL THERAPY OF LEUKEMIC STEM CELLS USING BONE MORPHOGENETIC PROTEIN AND TYROSINE KINASE INHIBITORS

Background:Chronic myeloid leukemia (CML) disease outcomes have improved since the introduction of tyrosine kinase inhibitors (TKIs), however a proportion of patients will have TKI resistance and TKIs are not curative in the majority of patients. Low‐levels of Bcr‐Abl transcripts persist and a major clinical aim is to understand mechanism underlying disease persistence and the role of leukemic stem cells (LSCs) in this. To date it has proved difficult to both understand how LSCs dominate and alter the niche, and to target LSCs with current therapies. Previous studies have highlighted the importance of the bone morphogenetic protein (BMP) pathway in CML LSCs with altered stem cell fate, persistence and the response to BMP antagonists affecting cell behavior with altered cell cycle, apoptosis and expansion (Laperrousaz, B. et al, Blood, 2013; Grockowiak, E. et al, Blood, 2017).Aims:To investigate the effect of BMP pathway inhibition in combination with TKIs in primary CML samples to improve insights and identify new potential therapeutic approaches.Methods:Primary CML CD34+cells were treated with Imatinib (IM) or Saracatinib (SC), a Src/Abl dual inhibitor, alone or in combination with BMP inhibitor Dorsomorphin (DOR). Additionally, cell cultures were supplemented with soluble BMP4 or co‐cultured with the human stromal cell line HS‐5. Apoptosis, cell cycle and proliferation were investigated by flow cytometry. RNA was harvested, cDNA generated and pre‐amplified prior to the performance of multiplex Real‐Time PCR (Fluidigm) on 89 lineage‐specific, cell cycle, proliferation, and survival genes. Furthermore, we investigated tyrosine kinase activity in cell lysates of primary CML patient samples post single or dual treatment of SC and DOR using a chip‐based microarray assay from PamGene containing 196 peptides. Kinase phosphorylation activities measured on peptides were further evaluated by relating them to kinases that are able to phosphorylate a particular site on the peptide sequence.Results:Combinatorial treatments targeting the BMP pathway together with TKIs display a synergistic mode of action. Increases in late apoptosis (p &lt; 0.0002), altered cell cycle (G2‐M, p = 0.0058), fewer cell divisions, and reduced numbers of CD34+cells were observed in dual treatments. BMP4 supplementation revealed similar differences whereas stroma co‐cultures displayed a protective effect. Furthermore, multiplex quantitative PCR has revealed significant deregulation of genes involved in cell cycle,apoptosis and differentiation, such as CDKN1a, CDKN2b, BCL6, GATA1 and SMAD5 showing differential expression in relation to response. Upstream kinase analysis using the PamGene platform, revealed kinases involved in cell growth, development, differentiation, apoptosis and cell‐cell adhesion. Amongst the top 20 potential differentially expressed kinases after SC treatment, were the expected members of the Src family kinases. DOR treatment revealed an up regulation of the largest known subfamily of receptor tyrosine kinases, the Ephrin receptors and Met, a single pass tyrosine kinase receptor that had already been identified as a potential therapy target in CML for IM resistant cells.Summary/Conclusion:Our results highlight the potential of a combined approach of targeting the BMP pathway with BMP antagonists or small inhibitors together with TKIs, which could open up new therapeutic possibilities. Future experiments will assess the potential of combinatorial treatment in a 3D bone marrow model, comprising magnetically levitated Mesenchymal Stem Cell spheroids embedded in medical‐grade collagen type I.

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy

AbstractAntileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry–based approach to identify naturally presented HLA class I– and class II–restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL– and ABL-BCR–derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell–based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

Differential Expression of Circadian Genes in Leukemia and a Possible Role for Sirt1 in Restoring the Circadian Clock in Chronic Myeloid Leukemia.

Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) – dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukaemia

BACKGROUND. Tyrosine Kinase Inhibitors (TKIs) are part of the successful clinical management of patients with Chronic Myeloid Leukaemia (CML). However, optimal clinical management of CML requires a robust, standardized laboratory assay used at key clinical milestones to ensure a successful outcome for patients on TKIs. Quantitative monitoring of %BCR-ABL1IS by reverse transcription quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to therapy and classification into prognostic subgroups. However, it can be challenging to perform in a reproducible manner. Reverse-Transcription Digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for patient stratification.AIM. In this study, we compared three different dPCR platforms and investigated whether they could be applied to a clinical setting to quantify BCR-ABL1 transcripts in CML patients.METHODS. BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. Three commercially available RT-dPCR platforms (QS3D, QX200 and RainDrop) were compared with the routinely used RT-qPCR platform using a modified version of the Europe Against Cancer (E.A.C.) assay.RESULTS. Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1% (R2 = 0.91, 0.93, 0.95 for QS3D, QX200, RainDrop, respectively)(Figure 1A). RT-dPCR quantification of the BCR-ABL1 transcript copy numbers however correlated well with RT-qPCR across all three platforms down to ≥0.1% (R2 = 0.85, 0.94, 0.92 for QS3D, QX200, RainDrop, respectively) (Figure 1B). Below this level, while the correlation was maintained, there was greater variation among instruments between RT-dPCR and RT-qPCR. Agreement between instruments was consistently higher for BCR-ABL1 copies than for ABL1 copies on the same patient set; thus changing the %BCR-ABL1 ratio values frequently varying by one order of magnitude when compared to RT-qPCR (R2 = 0.89, 0.92, 0.97 for QS3D, QX200, RainDrop, respectively) (Figure 1C). Furthermore, none of the platforms were able to substantially improve the sensitivity of quantification by RT-qPCR in relation to patients samples with %BCR-ABL1IS level <0.001%.When using the QS3D there was good correlation down to approximately 20 copies of BCR-ABL1 transcripts, below which a plateau was observed (Figure 2). For quantification of the lowest two CML categories, 0.001% and <0.001%, all three RT-dPCR instruments detected amplification of BCR-ABL1 targets (Figure 2). However, the comparison between these groups and the non-CML control groups showed a positive measurement in the absolute quantification of BCR-ABL1 in the negative control groups (false positive rate; FPR) that clearly affected the sensitivity of measurement in these two lowest CML categories (Figure 2). For the QX200 platform, the plateau was less pronounced than that observed with the QS3D. The FPR was visibly lower than those of the QS3D and Raindrop platforms. However, for the QX200, the lowest three CML categories (≤0.01%) were indistinguishable from the negative control patient groups (Figure 2). Only the Raindrop platform was able to measure % BCR-ABL1IS in the <0.001% range at a level higher than the FPR in the control patient groups (Figure 2). This was possibly related to the larger volume of cDNA added to each reaction (10 µl as opposed to 1 µl). However, in the CML patient samples, the Raindrop platform did not quantify any difference in the BCR-ABL1 transcripts in proportion to the disease level they were assigned (0.01%, 0.001% or <0.001%) (Figure 2).CONCLUSION. RT-dPCR was able to quantify low level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without calibration curve. Adaptions to the protocol to increase the amount of RNA measured, a redesign of the assay used and an alternative reference gene to ABL1 are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing. [Display omitted] [Display omitted] DisclosuresGriffiths:Affymetrix: Research Funding. Apperley:Bristol Myers Squibb: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Ariad: Honoraria, Speakers Bureau.

Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia Identifies Novel CD8+ and CD4+ T Cell-Epitopes for Immunotherapeutic Approaches

While the discovery of BCR-ABL and the respective tyrosine kinase inhibitors (TKI) resulted in a significant prolongation of patient survival rates, there still is no curative treatment for chronic myeloid leukemia (CML) except for allogeneic stem cell transplantation. The concept of T cell-based immunotherapy is a promising opportunity to eliminate residual leukemic cells, which might promote disease relapse after TKI discontinuation. As effective antigen-specific immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens, we have developed a mass spectrometry-based approach, which allows for the direct identification of naturally presented tumor-associated HLA ligands in hematological malignancies. In this study we used this approach to identify HLA class I and II CML-associated peptides as targets for T cell-based immunotherapy.Analysis of HLA class I ligandomes of primary CML cells (n=16) identified 8,291 HLA ligands representing 4,337 source proteins. Comparative ligandome profiling using a benign HLA class I database, which includes various healthy tissues (n=188, 65,949 HLA ligands, 14,030 source proteins) originating from peripheral blood, bone marrow, kidney, lung, liver, colon, spleen and others, revealed 38 CML-exclusive HLA class I ligands with frequencies ≥ 25% of CML patients. Because of the important indirect and direct roles of CD4+ T cells in anti-cancer immune responses, an optimal immunotherapy approach requires the inclusion of HLA class II epitopes. Hence we also analyzed the HLA class II ligandomes of primary CML cells (n=15, 2,822 HLA ligands, 794 source proteins). Comparative ligandome analysis (benign tissue, n=114, 54,149 HLA ligands, 8,584 source proteins) identified 44 CML-associated HLA class II ligands showing CML-exclusive representation in > 25% of the analyzed CML samples.To validate the immunogenicity of our HLA class I and II CML-associated peptides, we performed IFNγ- ELISPOT assays after 12-days of in vitro peptide stimulation. For HLA class II antigens, a panel of 4 peptides was implemented for stimulation of PBMCs obtained from CML patients and healthy volunteers (HV). The ELISPOT assay revealed peptide-specific immune recognition of 4/4 (100%) CML-exclusive peptides in CML patients. The frequencies of the detected immune responses ranged from 17% (4/23 patients) to 4% (1/23 patients) within the tested CML samples. These immune responses were mediated by functional CML patient-derived CD4+ T cells and strictly CML-directed, as no immune response against CML-associated peptides could be detected in HV (0/8).For HLA class I antigens, ELISPOT assays were performed using a panel of 8 peptides. Immune responses were only detected for 1/8 (13%) peptides with a low frequency of 6% (1/18 patients) of tested CML patient samples. A possible explanation for the observed weak immune response to our HLA class I CML-associated peptides compared to the immune responses shown for HLA class II peptides and for HLA class I peptides in other hematological malignancies (e.g. CLL (Kowalewski et. al. PNAS 2015)) might be an inhibition of CD8+ T cell-responses, that reportedly occurs upon TKI treatment of CML patients. To prove this hypothesis in our CML patient cohort (all patients included were under TKI treatment at the time of sample collection), we compared the ELISPOT positive controls (stimulated with a set of 5 Epstein-Barr viral peptides) of all analyzed CML samples with positive controls derived from HV and CLL samples. We could show a highly significant mean spot count reduction (per 100,000 cells) in CML samples (mean 74±16 spots, n=19) compared to HV (mean 241±24 spots, n=42, p<0.001, two-tailed t-test) or CLL (mean 218±16 spots, n=125, p=0.008) samples, confirming the general debilitated CD8+ T cell-response in CML patients under TKI treatment.To prove the immunogenicity of our HLA class I CML-associated peptides, we performed in vitro artificial antigen-presenting cell (aAPC)-based priming experiments of HV CD8+ cells. For one HLA-A*03-restricted peptide we observed tetramer-positive CD8+ populations with frequencies ranging from 0.12% to 1.41% of viable cells in 2/2 HVs so far.Taken together, these results are a first step towards a successful validation of these newly defined HLA class I and II CML-associated antigens as prime targets for further T cell-based immunotherapy approaches in CML patients. DisclosuresKowalewski:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.