Residual Chronic Myeloid Leukemia Cells Research Articles

High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation.

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

Effects of the STAMP-inhibitor asciminib on T cell activation and metabolic fitness compared to tyrosine kinase inhibition by imatinib, dasatinib, and nilotinib

T cell function is central to immune reconstitution and control of residual chronic myeloid leukemia (CML) cells after treatment initiation and is associated with achieving deep molecular response as a prerequisite for treatment-free remission, the ultimate therapeutic goal in CML. ATP-pocket-binding tyrosine kinase inhibitors (TKIs) like imatinib, dasatinib, and nilotinib are widely used for treating CML, but they have shown to inhibit T cell function as an “off-target” effect. Therefore, we tested asciminib, the first-in-class BCR::ABL1 fusion protein inhibitor specifically targeting the ABL myristoyl pocket (STAMP) and compared its effects on T cell function with imatinib, dasatinib, and nilotinib. Whereas all four TKIs inhibited the expression of the co-stimulatory protein CD28, the amino acid transporter CD98, proliferation, and secretion of pro-inflammatory cytokines IFNγ, IL-6, and IL-17A upon T cell stimulation, asciminib had less impact on PD-1, activation markers, and IL-2 secretion. T cells treated with asciminib and the other TKIs maintained their ability to mobilize their respiratory capacity and glycolytic reserve, which is an important surrogate for metabolic fitness and flexibility. Overall, we found milder inhibitory effects of asciminib on T cell activation, which might be beneficial for the immunological control of residual CML cells.

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Aurora A Kinase As a Promising Therapeutic Target in Philadelphia Chromosome Positive Leukemia Cells

Introduction: ABL tyrosine kinase inhibitor (TKI), imatinib and second-generation ABL TKIs, such as nilotinib and dasatinib have demonstrated the potency against chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (Ph+ALL) patients. However, resistance to ABL TKI can develop in CML patients due to BCR-ABL point mutations. Moreover, ABL TKIs do not eliminate the leukemia stem cells (LSCs). Residual CML cells are present in bone marrow microenvironment. These cells are contained within a niche in the bone marrow and are often impervious to current treatments. Aurora kinases are novel family of serine/threonine kinases that play a critical role in regulating many of the processes that are pivotal to mitosis. Aurora kinases comprises three members, designated aurora A, B, and C. Amplification of aurora kinase gene is oncogenic and has been observed in several cancers including hematologic malignancies. Therefore, new approach to the treatment for Ph-positive cells with aurora kinase inhibitors may improve the outcome of leukemia patients.Materials and methods: In this study, we investigated whether aurora kinase inhibitor could suppress Ph-positive leukemia cells including T315I mutation and primary samples.Results: One of aurora kinase A inhibitor, alisertib inhibits proliferation of leukemia cells. We found alisertib treatment inhibited the growth of Ph-positive leukemia cells in a dose dependent manner and phosphorylation of aurora kinase A was reduced. Alisertib inhibits proliferation in imatinib resistant cells including T315I mutation. In the cell cycle analysis, alisertib induces a G2/M phase arrest in Ph-positive cells. Intracellular ROS levels were also increased in a dose dependent manner. Combined treatment of Ph-positive cells with ABL tyrosine kinase inhibitors, imatinib or ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Apoptotic and Caspase 3/7 positive cells were also increased. Ph-positive cells were treated with alisertib and cellular senescence was determined by senescence- associated β-galactosidase (SA-β-gal) staining. Senescent cells were increased in a time and dose dependent manner. SA-β-gal positive cells were reduced by N-Acetyl-L-cysteine treatment. Small interfering RNA mediated knockdown of the Aurora A gene causes a proliferation defect and enhances ABL TKI activity in Ph-positive cells. We next examined tumor formation in mice model. We injected subcutaneously 1×107 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. We also found that co-treatment with ponatinib and alisertib increased mouse survival. Co-treatment with ponatinib and alisertib showed reduced mouse spleen weight and size. The treatments were well tolerated with no animal health concerns observed.Conclusion: The results of our study indicate that the aurora kinase inhibitor may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. DisclosuresTauchi:Otsuka pharmaceutical KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau. Ohyashiki:Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kyowa Hakko Kirin KK: Honoraria; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; MSD KK: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Chugai pharmaceutical KK: Research Funding; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; MSD KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding.

Treatment-free remission and immunity in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is caused by the reciprocal translocation t(9;22)(q34;q11), resulting in the BCR-ABL1 fusion gene. BCR-ABL1 tyrosine kinase inhibitors (TKIs) improve overall survival in patients with chronic phase CML (CML-CP). Approximately half of the patients who achieve a durable deep molecular response can achieve sustained treatment-free remission (TFR) after TKI discontinuation; thus TFR is now a therapeutic goal for most patients with CML-CP. Sensitive BCL-ABL1 transcript detection methods reveal that evidence of residual CML cells remains in patients who achieve sustained TFR, indicating that the host immune system protects against CML relapse. The human immune system is composed of innate and adaptive arms. Natural killer cells are major components of the innate immune system, while T cells are major components of the adaptive immune system. Myeloid-derived suppressor cells and regulatory T cells, both suppressors of the immune response, have important roles in the regulation of CML. Here, we review the current understanding of the immune response in CML, especially in TFR.

Lineage of measurable residual disease in patients with chronic myeloid leukemia in treatment-free remission.

Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10-4.9 vs 10-5.7; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR.

Increased Tumour Burden over a 36 Month Period in Chronic Myeloid Leukemia Patients Following Imatinib Discontinuation: Role of Digital PCR

Le Coutre: Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau. Elena:Novartis: Consultancy; Pfizer: Consultancy. Assouline:Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria. Stagno:Incyte: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Kim:Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Il-Yang co.: Research Funding. Fava:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Gambacorti-Passerini:Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

PF414 DNA‐BASED BCR‐ABL1 ANALYSIS PROVIDES A “TRAFFIC LIGHT” STRATIFICATION MODEL FOR CHRONIC MYELOID LEUKEMIA WITH IMPLICATIONS FOR TREATMENT FREE REMISSION

Background:Treatment free remission (TFR) or molecular relapse free survival (MRFS) have become important therapeutic aims for patients with chronic myeloid leukemia (CML). This aim can be realised through achievement of a sustained deep molecular response (DMR).Aims:In our previous work (Machova Polakova K, EHA 2018) we showed that a BCR‐ABL1 DNA‐based approach for residual disease monitoring may be superior to mRNA analyses in a significant number of peripheral blood samples. In this collaborative EUTOS study we aimed to assess whether DNA PCR provided better prediction of TFR.Methods:Consecutive samples before and after TKI cessation were available for 54 CML patients, of whom 16 were enrolled in the EURO‐SKI study. Thirty‐five patients, who fulfilled criteria but could not be involved in the EURO‐SKI trial, gave consent for an intermittent regimen (INTReg) of TKI administration every second month. The MRFS rate of INTReg patients was comparable to EURO‐SKI and other TKI stopping trials in later time points (39% at 20 months and 32% at 70 months after TKI cessation). Patient‐specific genomic BCR‐ABL1 fusions were characterized by next generation sequencing and measurable residual disease was compared using DNA and mRNA based digital droplet (dd) PCR. The sensitivity of standard DNA and mRNA analysis of peripheral blood samples were comparable, varying between MR4‐MR5.Results:DNA and mRNA BCR‐ABL1 levels from consecutive analysis were available for 33/54 patients (altogether 945 samples; median 30 samples per patient; range 11–44). Analysis of these 33 cases enabled a “traffic light” stratification according to BCR‐ABL1 DNA and mRNA status before TKI cessation (Figure 1); “Green” group ‐ continually double negative (n = 4), “Yellow” group ‐ continually DNApos/RNAneg (n = 13) and “Red” group ‐ double positive (n = 16). All 4 patients from the Green group sustained in MRFS with continuous double negative status. Two of these patients gave consent for bone marrow (BM) aspiration at the time of TKI cessation. Both BM samples were double negative when analysed by ddPCR with enhanced sensitivity of MR6‐MR6.5 by performing multiple replicates. Molecular relapse (loss of MMR) occurred in 12/16 patients of the Red group. In the Yellow group, 8/13 patients survive without molecular relapse after TKI cessation (median 39.8 months; range 19.1–82.2); in 5/8 patients, the BCR‐ABL1 expression became detectable, however, only at DMR levels, i.e. MR4 or lower. BCR‐ABL1 expression remained undetectable in 3/8 patients after TKI cessation (median 27.3 months; range 19.1–53.6). BM was collected in 2/3 patients at the time of TKI cessation; in both cases, DNA assays detected residual CML cells despite the absence of BCR‐ABL1 expression.Summary/Conclusion:BCR‐ABL1 DNA analysis allows clinically relevant stratification of CML patients in DMR before complete or intermittent treatment cessation. Our approach identifies truly BCR‐ABL1 negative patients that are likely to benefit from TKI cessation. A biologically interesting Yellow group represents patients with detectable CML cells but with undetectable BCR‐ABL1 expression on TKI therapy. Although BCR‐ABL1 expression became detectable after TKI cessation in many of these cases, a high proportion (62%) achieved MRFS. Molecular relapse occurred in a high proportion (76%) of double positive patients. Currently we are validating the “traffic light” stratification scheme on the remaining 21 patients from our cohort.imageSupport: EUTOS2018, MZCR 00023736, AZV 16–30186A

ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells.

SummaryProperties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.

Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia (CML)—Towards Peptide Based Immunotherapy

Despite the success of targeted therapy with tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable. Immunotherapy, and in particular multi-peptide vaccination, may be a promising approach to eliminate residual CML cells. As of now, a multitude of potential vaccine targets have been proposed based on reverse immunology and functional genomic approaches focusing either on BCR-ABL junction peptides, which represent CML-specific neo-antigens, or on aberrantly expressed self-proteins such as WT-1, PR and hTERT. However, the results of clinical studies employing such antigens have so far not been encouraging. This might in part be due to the inherent limitations of the above mentioned approaches: evidence of natural presentation of the predicted epitopes is lacking and the correlation of transcript abundance and HLA restricted presentation of the corresponding gene product has been shown to be skewed. Modern mass spectrometry, on the other hand, enables the comprehensive analysis of the entirety of naturally presented HLA ligands on tissues of interest, termed the HLA ligandome.Here we implemented this direct approach and comparatively mapped the HLA ligandome landscape of 16 primary CML samples and 40 healthy volunteer (HV) controls (30 blood and 10 bone marrow samples). We identified more than 30,000 different naturally presented HLA class I ligands representing ~10,000 source proteins. Regression analysis suggests source protein identifications on CML (4,337 different proteins) to attain >95% of maximum achievable coverage with the implemented analytical setup. Based on this extensive dataset, we investigated the HLA restricted presentation of established CML-associated/specific antigens and applied a novel approach defining tumor-associated antigens strictly based on exclusive and frequent representation in CML ligandomes. Strikingly, we found the vast majority of previously described antigens including wild-type BCR protein (6% CML, 5% HV), Myeloperoxidase (56% CML, 15% HV) and Proteinase 3 (38% CML, 11% HV) to be (also) represented on normal PBMC or BMNC. No evidence of naturally presented BCR-ABL junction peptides was found. However, we identified a panel of 7 LiTAAs (ligandome-derived tumor-associated antigens) represented by 16 different HLA ligands, showing CML-exclusive representation in ≥25% of CML patient ligandomes.As CD4+ T cells mediate important indirect and direct effects in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 15 CML patients and 18 HV (13 blood and 5 bone marrow samples), identifying more than 9,000 different naturally presented HLA class II ligands (1,900 source proteins). Applying the same antigen-ranking strategy as described for HLA class I, we identified 7 additional HLA class II LiTAAs represented by 50 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of CML-exclusive source proteins revealed 6 proteins to be represented both in HLA class I and II ligandomes. Notably, for Galectin-1, which shows CML-exclusive representation in 19% of HLA class I and 13% of HLA class II ligandomes, one of the HLA class II ligands was found to contain a complete, embedded HLA class I peptide. Such naturally presented embedded HLA ligands might present optimal vaccine candidates that are recognized by both, CD4+ and CD8+ T cells.Functional analysis of the here defined HLA class I and II LiTAPs with regard to induction of T cell responses is presently ongoing and serves to validate them as prime targets for the development of an off-the-shelf peptide vaccination in CML patients. DisclosuresNo relevant conflicts of interest to declare.

Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells.

BackgroundThe ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2.MethodsWe investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).ResultsImatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.ConclusionsThese results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

HLA Ligandome Analysis Of Chronic Myeloid Leukemia (CML), Revealed Novel Tumor Associated Antigens For Peptide Based Immunotherapy

Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Specific T-cell based immunotherapies for patients with CML might be able to eliminate these residual CML cells. For this goal the identification of tumor associated HLA-presented peptides, which are able to induce a tumor-specific T cell response, is indispensable. However, only few tumor associated antigens for CML are described till date. Thus the aim of this study was to identify for the first time naturally processed and presented HLA ligands from the cell surface of primary CML cells. HLA class I ligands from primary CML cells as well as from bone marrow mononuclear cell (BMNCs) and peripheral blood PBMCs of healthy donors were analyzed using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry (LC-MS/MS). LC-MS/MS peptide analysis provided qualitative and semi-quantitative information regarding the composition of the respective ligandomes. Comparative analysis of malignant and benign samples served to identify ligandome-derived tumor associated antigens (LiTAAs) and to select peptide vaccine candidates. So far 10 CML patients (8 chronic phase, 2 accelerated phase) and 30 healthy donors were analyzed within this study. We were able to identify a total of more than 8200 CML derived HLA ligands representing >4500 different source proteins, of which 734 were exclusively represented in CML, but not in healthy PBMCs/BMNCs. 55 of these CML exclusive antigens are presented on 3 or more of all examined CML patients, representing, as broadly represented LiTAAs, promising targets for peptide vaccination. For the first time, previously predicted CML tumor associated antigens for example Myeloperoxidase (10 peptide sequences on 7 CML patients) and Proteinase 3 (5 peptide sequences on 4 CML patients) were here confirmed by direct elution from primary CML cells, which also served to validate our methodological approach. Notably, beyond that we also identified a vast array of novel proteins (e.g. Carcinoembryonic antigen-related cell adhesion molecule 8, CEACAM8; Matrix metallopeptidase 8, MMP8; intracellular adhesion molecule 3, ICAM3) that are broadly and exclusively represented in the ligandome of CML cells.By providing for the first time HLA class I tumor associated antigens, directly obtained from the HLA ligandomes of CML patients, this study may pave the way for the future development of a multipeptide-based immunotherapy approach to eradicate residual CML cells after conventional TKI therapy. Disclosures:No relevant conflicts of interest to declare.

Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells

Abstract 3737ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures:No relevant conflicts of interest to declare.

GenomicBCR‐ABL1breakpoints in pediatric chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a rare disease in children and adolescents and various aspects-from molecular genesis to therapy regimen-have been taken over from studies on the more prevalent adult CML. However, differences have been observed between malignancies with identical underlying chromosomal translocations, but occurring at different age groups, suggesting some diversity in the mechanisms of formation and leukemogenesis. A multiplex long-range PCR-based assay was developed to allow fast and reliable amplification of patient-specific BCR-ABL1 fusion sequences from genomic DNA. The localization of breakpoints was analyzed with respect to distribution within the breakpoint cluster regions, sequence features, and association to repetitive elements or motifs associated with DNA recombination. The genomic fusion sites of 59 pediatric CML patients showed a bimodal breakpoint distribution in BCR that was different from the distribution in adult CML cases, but with similarities to BCR-ABL1-positive, acute lymphoblastic leukemia in adults. BCR breakpoints were found more frequently positioned within, or close to, Alu repeats than would be expected based on their overall sequence proportion. Technical aspects of the highly sensitive DNA-based quantification of residual CML cells by specific fusion sequence during tyrosine kinase inhibitor therapy are exemplified in a subcohort of pediatric CML patients.

Abstract 2682: New diagnostic and therapeutic target KU-MEL9 for chronic myeloid leukemia (CML)

Abstract Overall survival of patients with chronic myeloid leukemia (CML) has dramatically changed over past ten years by the introduction of targeted therapy with tyrosine kinase inhibitors (TKIs). However, it has been reported that more than half of patients eventually relapse after the cessation of TKIs, suggesting that CML cells including leukemic stem cell (LSC) cannot be completely eradicated by TKIs. To this end, the identification of targets expressed in LSC is crucial for developing highly effective therapies for CML. In this study, we evaluated the gene expression of cancer testis antigen KU-MEL9, which was found in melanoma research, in a variety of human cell lines. This gene was highly expressed in several cell lines of hematological malignancies including CML and multiple myeloma (MM). We then investigated the gene expression of KU-MEL9 in bone marrow samples from 20 CML patients by standard RT-PCR method. Gene expression of KU-MEL9 were found in 17 patients (85%), and correlated with the expression of BCR-ABL fusion gene in most of the cases. On the other hand, 6 samples from normal donors didn't express this gene. In 2 CML patients who achieved a molecular remission with imatinib, the expression of KU-MEL9 gene was still observed, although BCR-ABL gene was not detected. It suggested that KU-MEL9 was expressed in residual CML cells including LSC and could be a more sensitive biomarker for CML patients. We also examined the possibility of KU-MEL9 being as a target of cytotoxic T cells (CTL) for immunotherapy to eradicate remaining LSCs after TKI treatment. Although this study had limited number of patient sample, KU-MEL9 may be a useful target for diagnosis and treatment for CML patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2682. doi:1538-7445.AM2012-2682

Imatinib-Loaded Polyelectrolyte Microcapsules for Sustained Targeting of BCR-ABL + Leukemia Stem Cells

The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib

AbstractPrimitive quiescent CD34+ chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34+ cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34+ populations. Quiescent CD34+ cells from CML patients were less susceptible than their cycling CD34+ and CD34− counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34+ CML cells had higher surface expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34+ CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell–mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

Upregulation of CXCR4 Is Essential for the Migration and Survival of Quiescent CML Progenitor Cells in the Bone Marrow Microenvironment after Imatinib Treatment.

Chronic myeloid leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow (BM) stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, resulting in cell migration defects in CML. We proposed that the tyrosine kinase inhibitors imatinib or INNO-406 may restore CXCR4 expression and cause the migration of CML cells to BM microenvironment niches which results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib or INNO-406 increased CXCR4 expression and migration. Imatinib induced a G0/1 cell cycle block in CML cells, which was further enhanced in a bone marrow-derived stromal cell (MSC) co-culture system. Quiescent KBM5 cells migrating to stromal cells were resistant to Imatinib-induced cell death. These anti-apoptotic effects were abrogated by inhibition of CXCR4 (AMD3465) or Integrin-linked kinase (QLT0267). BM CD34+ cells from newly diagnosed (n=15) or blast crisis CML patients (n=5) expressed markedly lower CXCR4 levels compared with normal CD34+ BM progenitor cells (n=12) (p<0.05), and was associated with deficient migration of CD34+ CML cells. A longitudinal analysis of peripheral blood samples from CML patients in blast crisis treated with imatinib (n=5) revealed induction of CXCR4 in peripheral blood CD34+ progenitor cells within the first few days after initiation of treatment, which coincided with decreased WBC and absolute blast counts. After prolonged (3–10 day) exposure to imatinib, however, CXCR4 levels of CD34+ PB CML cells decreased, frequently below the starting levels. CXCR4 immunostaining on blasts/immature cells in BM biopsy sections from patients demonstrated that 5 of 7 patient samples were CXCR4-negative prior to imatinib therapy; 4 of these 5 became CXCR4 positive after imatinib treatment. While patients in blast-phase had short-lived hematologic responses to imatinib, Ph+ clone remained predominant in the bone marrow, and all patients ultimately relapsed with recurrence of blasts in the BM. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to BM stroma, causing G0/1 cell-cycle arrest and enhancing the survival of quiescent CML progenitor cells within bone marrow microenvironment. This provides the rationale for interfering with the protective effects of BM stroma cells by inhibiting CXCR4 and/or integrin signaling, which could be of benefit in eradicating residual quiescent CML cells.

Chronic myeloid leukemia cells express tumor-associated antigens eliciting specific CD8 + T-cell responses and are lacking costimulatory molecules

Specific immunotherapies for patients with chronic myeloid leukemia (CML) might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft-versus-leukemia effect after allogeneic stem cell transplantation. Here, we investigated the mRNA expression and T-cell recognition of tumor-associated antigens or leukemia-associated antigens (LAAs) in 34 patients with CML. Several LAAs are expressed in CML and therefore are candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%), and WT1 (53%), but not BAGE, MAGE-A1, SSX2, or NY-ESO-1. The frequency of mRNA expression of RHAMM/CD168, Proteinase3, and PRAME was higher in acceleration phase and blast crisis. In flow cytometry, CD34+ progenitor cells typed positive for HLA molecules but were deficient for CD40, CD80, CD83, and CD86. However, RHAMM/CD168 R3-peptide (ILSLELMKL)-specific T-cell responses in CML patients were demonstrated by ELISPOT analysis and specific lysis of RHAMM/CD168 R3-pulsed T2 cells and CD34+ CML cells in chromium-51 release assays. RHAMM-R3-specific T cells could be phenotyped as CD8+R3*tetramer+CD45RA+CCR7-CD27- early effector T cells by tetramer staining. Therefore, vaccination strategies inducing such RHAMM-R3-directed effector T cells might be a promising approach to enhance specific immune responses against CML cells.

Chronic Myeloid Leukemia (CML) Cells Express Tumor Associated Antigens Eliciting Specific CD8+ T Cell Responses Despite of Deficient Expression of Costimulatory Molecules.

Specific immunotherapies for CML patients targeting T cell antigens might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft versus leukemia effect after allogeneic stem cell transplantation without aggravating the graft versus host disease. However, for effective specific immunotherapies in CML, extended studies on the expression, the function and the immunology of leukemia-associated antigens (LAAs) and on the LAA presentation by leukemia cells are required.Here, we investigated on expression and functional aspects of tumor/leukemia-associated antigens (TAAs/LAAs) in CML. Several LAAs are expressed and are therefore candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%) and WT1 (53%), but not BAGE, MAGE-A1, SSX2 or NY-ESO-1. The expression of LAAs varied according to leukocyte subsets and the expression of RHAMM/CD168, Proteinase3 and PRAME was upregulated in accelerated phase and blast crisis. Quantitative expression of several TAAs/LAAs correlated with the clinical course. In flow cytometry, CD34+ CML progenitor cells typed positive for HLA-molecules, but were deficient for CD40, CD80, CD83 and CD86. This lack of costimulatory molecules might constitute a tumor escape mechanism. However, RHAMM/CD168-R3-specific T cell responses were demonstrated by ELISPOT analysis and specific lysis of R3-peptide pulsed T2 cells in chromium-51 release assays. These CD8+ cells could be phenotyped as CCR7-CD27-CD45RA+ early effector T cells by tetramer staining. In conclusion, several LAAs are expressed in CML and are therefore candidate structures for specific immunotherapies. CML progenitor cells express HLA-molecules, but lack costimulatory molecules. However, as CML patients show RHAMM/CD168-R3-specific early effector T cells, peptide vaccination might be therefore a promising approach to enhance specific immune responses in CML.