Chronic Myeloid Leukemia Patients In Remission Research Articles

Myelofibrosis at diagnosis is associated with the failure of treatment-free remission in CML patients.

The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor associating with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR::ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Analysis of human CML-vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, whereas this was only the case in 25% of patients with achieved TFR, further supporting the link between fibrosis and LSC persistence.

Integrated High-Throughput Drug Profiling and CRISPR Screening Identify Novel Pathway Vulnerabilities of Leukemic Stem Cells in Chronic Myeloid Leukemia

Despite BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) have dramatically improved the management of chronic myeloid leukemia (CML) patients, TKIs fail to eradicate leukemic stem cells (LSCs). CML-LSCs are recognized as BCR-ABL-kinase activity independent and responsible for TKI-resistance and relapse upon treatment discontinuation. Systematic mapping of druggable signaling pathways enables the identification of novel CML-LSCs effectively targeting drugs, which is critical to enhance the achievement of sustained treatment free remission in CML patients. We implemented ex-vivo drug testing of 80 targeted drugs and 18 drug combinations across 10 000-fold concentration, to evaluate drug sensitivity profiles of primary CML-LSCs and progenitors (CD34+ cells) from twenty patients in addition to CD34+ cells from three healthy donors. We identified novel drug vulnerabilities of CML-LSCs including VEGFR-, CDK2/WEE-, MDM2-, and PI3K/mTOR- inhibitors. Combination of imatinib with idasanutlin, venetoclax, AZD1775 and dexamethasone led to synergistic inhibition of CML-LSCs. To delineate the phenotype-specific sensitivity profile of CML CD34+ cells, we applied ex-vivo flowcytometry-based drug screening approach to bone marrow samples from ten CML patients using a custom library of 20 drugs. We identified specific sensitivity of CML CD34+CD38- cells to mepacrine, RG7112, idasanutlin, and navitoclax. In particular, mepacrine showed striking ability to induce differentiation of CML-LSCs, which was further validated in a separate cohort of primary CML samples and cell lines. To characterize the mechanisms of action of the discovered drugs, we applied genome-scale CRISPR screening in CML cells to identify potential sensitizing and resistance targets for five selected drugs. Imatinib, dasatinib, and tivozanib showed a similar profile of resistance and sensitizing genes, while AZD1775 and mepacrine had unique profiles. We identified knockout targets that confer BCR-ABL1-independent resistance common to different TKIs including mediator complex (MED23, MED26, CCNC), and apoptosis related (APAF1) genes in addition to other genes with uncharacterized role in CML, such as KCTD5 and EIF2AK1. On the other hand, knockout of certain genes was associated with increased sensitivity to specific drugs such as IGF1R-dasatinib and SPNS1-mepacrine associations. KCTD5 was consistently identified as the top resistance gene to all tested TKIs, and therefore we next investigated the potential role of KCTD5 in CML pathogenesis and TKI resistance. In RNA sequencing data, KCTD5 was significantly downregulated in CML-CD34+ cells compared to healthy counterparts. Using co-immunoprecipitation, we revealed that KCTD5 directly interacts with BCR-ABL and mediate TKI-induced ubiquitination by E3 ligase, as shown by WB assay, that can destine BCR-ABL for subsequent degradation. Knockout of KCTD5 was associated with decreased ubiquitination of BCR-ABL and resistance to imatinib in CML cell lines and primary CML-CD34+ cells. Furthermore, combination of TKI with deubiquitinase inhibitors induced enhanced TKI sensitivity in KCTD5-KO cells. In conclusion, we discovered novel mechanisms of primary TKI-resistance and drug vulnerabilities of CML-LSCs, which potentially enable eradication of LSCs in CML patients and hence pave the way for the achievement of functional cure in CML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Loss of G0S2 in Kinase-Independent TKI Resistance and Blastic Transformation of CML

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic condition. Despite impressive results, resistance is a clinical problem, and TKIs do not target the CML leukemic stem cell (LSC) population, which is independent of BCR-ABL1 kinase activity. To identify mechanisms associated with BCR-ABL1 kinase-independent TKI resistance, we compared the transcriptional profile of TKI-naïve CD34+ cells from newly diagnosed CML patients who subsequently either responded (n=41) or did not respond (n=18) to imatinib over a twelve-month period (McWeeney et al. Blood 2010). The transcriptional profile of imatinib resistance in non-responders revealed striking overlap with reported signatures of CML blastic transformation and also implicated pathways involved in LSC survival. These findings suggest that similar biological mechanisms may be driving three different scenarios of BCR-ABL1-independent resistance: 1) primary kinase-independent TKI resistance, 2) transformation of CML from the chronic phase (CP-CML) to the blastic phase (BP-CML), and 3) persistence of residual leukemia in TKI-responsive patients.Among the genes most profoundly downregulated in patients destined to fail imatinib therapy was G0S2 (>3-fold, p<0.02), encoding a small protein that regulates multiple cellular functions, including apoptosis, quiescence, lipid metabolism, and oxidative phosphorylation. We compared G0S2 mRNA expression in CD34+ cells from normal cord blood (n=5) and from patient specimens lacking BCR-ABL1 kinase domain mutations, including newly diagnosed CP-CML (n=6), TKI-resistant CP-CML (n=2), and BP-CML (n=5). G0S2 was 4-fold downregulated in newly diagnosed CP-CML compared to normal cord blood CD34+ cells (p<0.01), and further downregulated by >3-fold in TKI-resistant samples and BP-CML (p<0.01), consistent with published microarray data (McWeeney et al. Blood 2010). Retrospective analysis of survival data for patients whose samples were evaluated in the transcriptional profile revealed that low G0S2 expression correlated with a shorter overall survival (n=35, p=0.036). Consistently, G0S2 mRNA and protein were downregulated in TKI-resistant K562R cells lacking BCR-ABL1 kinase domain mutations compared to TKI-sensitive controls. Ectopic expression of Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation and increased apoptosis of TKI-sensitive and TKI-resistant K562 cells, and also CD34+ cells from CP-CML (n=5, p<0.05) and BP-CML (n=3, p<0.05) patients, having a more pronounced effect in BP-CML in the presence of imatinib. G0S2-Flag did not affect colony formation or apoptosis of normal cord blood CD34+ cells (n=2), which already express high levels of G0S2. Importantly, qRT-PCR revealed that G0S2 mRNA was significantly lower in primitive CD34+38- stem cells compared to more committed CD34+38+ progenitor cells in CP-CML (n=6, p<0.01) but not normal cord blood CD34+ cells (n=4, p=ns). These data suggest that loss of G0S2 plays a role not only in primary TKI resistance and blastic transformation of CML, but also in TKI resistance of the CML LSC. Consistently, CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions.Altogether, these findings suggest that G0S2 plays a role in regulating leukemic stem and progenitor cell survival and TKI response in vitro, and that restoring G0S2 expression combined with BCR-ABL1 inhibition may be a novel strategy to induce treatment-free remission in CML patients with kinase-independent TKI resistance and BP-CML, or to eradicate the CML LSC population. Future studies will assess the functional role of G0S2 in LSC survival and BCR-ABL1 leukemogenesis in vivo, and determine the mechanism by which G0S2 is downregulated in TKI resistance. DisclosuresDeininger:BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Celgene: Research Funding; Gilead: Research Funding; ARIAD: Consultancy.

Opportunities of Chronic Myeloid Leukemia Treatment with Reduced Doses of Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitor (TKI) therapy results in deep molecular response (MR) in 60-70 % of chronic myeloid leukemia (CML) patients. However, despite high efficacy of TKIs, many patients experience drug toxicity during the treatment. According to clinical studies, the probability of sustaining off-treatment remission in CML patients with deep MR is about 40-60 %. Great attention has recently been paid to personalized therapy of chronic phase CML. It consists in TKI dose modification to reduce or prevent adverse events. Major retrospective studies proved that in patients with optimal response TKI reduced doses can be considered safe from the point of view of sustaining major and deep MRs achieved with standard TKI doses. Also, prospective clinical trials deal with the follow-up using TKI reduced doses as pre-withdrawal period. But up to now, the results of only 4 of such studies have been available. To take a closer look at long-term follow-up of CML patients receiving reduced doses of TKIs, prospective clinical trials need to be carried out. The present article reviews the results of main studies dealing with management of CML patients treated with TKI reduced doses.

Efficacy of tyrosine kinase inhibitors on a mouse chronic myeloid leukemia model and chronic myeloid leukemia stem cells

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by constitutively active BCR-ABL1 tyrosine kinase resulting from the t(9;22) Philadelphia translocation. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), is a revolutionary molecular target inhibitor for CML. However, leukemic stem cells (LSCs) eventually become resistant to imatinib and thereby cause relapse. The next-generation BCR-ABL1 TKI dasatinib is also unable to eliminate CML LSCs. On the other hand, the third-generation BCR-ABL1 TKI ponatinib is not well studied in terms of its efficacy on CML LSCs. Here, we evaluate the efficacy of ponatinib against CML LSC-containing lin-Sca-1+c-Kit+ (LSK) cells using a mouse CML-like model. To this end, we compared the efficacy of imatinib, dasatinib, and ponatinib on CML LSK cells and showed that ponatinib is more effective at eliminating CML LSK cells. Our results suggest that ponatinib could be potentially useful for achieving treatment-free remission in CML patients.

Treatment of Chronic Myeloid Leukemia According to Current Guidelines: The Results of the Pilot Prospective Study “Early Induction Therapy and Monitoring”

Background. Current clinical guidelines on diagnosis and treatment of chronic myeloid leukemia (CML) define indications for substitution of first-line tyrosine kinase inhibitor (TKI) at therapy failure during different phases of disease progression. Aim. To assess the efficacy of CML treatment with implementing the protocol of timely monitoring and switching to another TKI. Materials &amp; Methods. Patients were included into pilot prospective study РИТМ during 5 years. Data on 100 CML patients were analyzed. Therapy and monitoring were conducted according to the Federal clinical guidelines on CML diagnosis and therapy, 2013. Results. Median follow-up after initiation of treatment was 46 months (range 12-74). Imatinib mesylate was administered as first-line therapy to 91 (91 %) patients, 9 (9 %) patients received 2nd generation TKI (TKI2). Therapy failure was registered in 31 (31 %) patients; 26 (84 %) of them were switched to TKI2. At the time of analysis 95 (95 %) patients were followed-up. Cumulative incidence of CML-associated mortality was 2 %. By the fifth year of follow-up cumulative probability of complete cytogenetic, major and deep molecular responses was 93 %, 88 % and 66 %, respectively. Conclusion. CML treatment according to current guidelines yields the results comparable with those achieved by first-line TKI2 therapy. This approach reduces CML treatment costs and lowers the risk of TKI2-associated adverse events. Due to a high rate of deep molecular response the proportion of CML patients in remission without treatment can be increased in the future.

CXorf48 is a potential therapeutic target for achieving treatment-free remission in CML patients

Although the introduction of tyrosine kinase inhibitors (TKIs) has improved overall survival of patients with chronic myeloid leukemia (CML), about half of the patients eventually relapse after cessation of TKIs. In contrast, the remainder of the patients maintain molecular remission without TKIs, indicating that the patients’ immune system could control proliferation of TKI-resistant leukemic stem cells (LSCs). However, the precise mechanism of immunity against CML-LSCs is not fully understood. We have identified a novel immune target, CXorf48, expressed in LSCs of CML patients. Cytotoxic T cells (CTLs) induced by the epitope peptide derived from CXorf48 recognized CD34+CD38− cells obtained from the bone marrow of CML patients. We detected CXorf48-specific CTLs in the peripheral blood mononuclear cells from CML patients who have discontinued imatinib after maintaining complete molecular remission for more than 2 years. Significantly, the relapse rate of CXorf48-specific CTL-negative patients was 63.6%, compared to 0% in CXorf48-specific CTL-positive patients. These results indicate that CXorf48 could be a promising therapeutic target of LSCs for immunotherapy to obtain durable treatment-free remission in CML patients.

DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4

Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Targeting of B-Cell Receptor Signaling Pathways

Abstract SCI-27Targeted therapy in hematologic malignancies has achieved significant therapeutic success when relatively selective inhibition is attainable to a target dispensable to the majority of normal cells. The best appreciated example of this is imatinib in chronic myeloid leukemia (CML), in which a single translocation forms a fusion protein involving the tyrosine kinase ABL that molecularly defines the disease. Kinase inhibition of ABL in this setting by imatinib (or other second- and third-generation kinase inhibitors) promotes durable, long-term remission in CML patients. The great majority of B-cell malignancies lack a characteristic translocation, activating mutation, or other aberration that facilitates such targeted therapy as employed in CML. Nonetheless, a common global signaling pathway involving the B-cell receptor (BCR) pathway has emerged as one that might be important to the control of these diseases. Furthermore, mouse models of select kinase targets (phosphoinositide-3 kinase p110 isoform-delta and Bruton's tyrosine kinase [BTK]) have shown that these are dispensable relative to long-term survival. Indeed, knockout or mutation of BTK and p110 delta each have a modest phenotype outside of diminished B-cell development and function. Based upon these preclinical observations, several molecules targeting BCR signaling have come forward to the clinic with exciting results across a wide range of B-cell malignancies. GS-1101 is a selective phosphoinositide-3 kinase p100 delta inhibitor with a very favorable toxicity profile that has shown promising clinical activity in low-grade lymphoma and chronic lymphocytic leukemia (CLL). The toxicity of this orally administered agent is quite modest in the majority of patients and allows sustained continuous dosing. Similarly, ibrutinib is an irreversible inhibitor of BTK and has shown promising clinical potential in an even broader range of B-cell malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, low-grade lymphoma, and CLL. Toxicity with ibrutinib has also been modest, allowing long-term continuous dosing. Notably, each of these agents also produces an atypical mobilization of malignant lymphocytes into the blood soon after treatment. This treatment lymphocytosis is BCR-target-related due to diminished CXCR4/SDF-1 interface between tumor cells and stromal cells in the bone marrow, with egress of these cells to the blood. GS-1101 and ibrutinib are now entering phase III studies for regulatory approval and offer great potential to change the treatment paradigm of both CLL and B-cell non-Hodgkin lymphoma (NHL). Questions moving forward with these agents will include molecular predictors of response, feasibility and efficacy of combining with other effective therapies, and mechanisms of resistance. The scientific session presentation will provide an overview of the most promising BCR signaling agents in CLL and NHL clinical trials. Disclosures:No relevant conflicts of interest to declare.

Chronic Myeloid Leukemia Patients in Prolonged Remission following Interferon-α Monotherapy Have Distinct Cytokine and Oligoclonal Lymphocyte Profile

Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×109/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ+ T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ+ T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.

Clonal Hematopoiesis in Philadelphia Chromosome-Negative Bone Marrow Cells of Chronic Myeloid Leukemia Patients Receiving Tyrosine Kinase Inhibitors

Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.

HTERT, the Catalytic Component of Telomerase, Is Down-Regulated in the Hematopoietic Stem Cells of Patients with Chronic Myeloid Leukemia.

Telomere shortening and increased telomerase activity is associated with disease progression in chronic myeloid leukemia (CML). In order to gain insight into the biology of telomerase and its regulation in CML we have evaluated the expression levels of the components of telomerase, its regulators, and several telomeric associated proteins in the hematopoietic stem cells (HSC)/leukemic blast cells of 21 patients with this disorder, as compared to the HSC population of a group of healthy individuals, using quantitative real time PCR. In this study we have made the surprising observation that hTERT, the catalytic component of telomerase, is down-regulated in the CD34+/blast cells of the majority of CML patients in chronic phase (CP) (7 out of 9, p=0.0339) relative to CD34+ cells from normal controls. Furthermore hTERT was found to be significantly down-regulated in 2 out of 3 patients in accelerated phase (AP) and in 9 out of 9 patients in blast crisis (BC) relative to normal controls (p=0.0003). The expression levels of hTR, the telomerase RNA template, and the telomeric- associated proteins TEP1, TRF1, TRF2 and tankyrase were all up-regulated in the CD34+/blast cells of the majority of the CML patients in CP and AP, relative to normal control CD34+ cells. With the exception of TEP1, the expression levels of all these factors were highest in CP CML and, in the majority of patients, decreased during disease progression. We speculate that the up-regulation of tankyrase (positive regulator of telomere length) and TRF2 (protects critically shortened telomeres) may contribute to telomere maintenance in CML. The expression levels of hTERT and c-myc, a positive regulator of hTERT expression, correlate in all stages of CML (R2=0.623). The expression levels of c-myc decreased with disease progression, falling below the levels observed in normal CD34+ cells in the majority of patients in BC. The expression levels of hTERT were increased in the CD34+ cells of three additional CP CML patients in remission following successful treatment with imatinib, relative to both untreated CP CML patients (p=0.0126) and to normal individuals, reflecting a restoration of haematopoiesis towards normal. In CML the turnover of all types of Ph+ stem and progenitor cells is known to be increased. We suggest that the reduced (and presumably inadequate) expression levels of hTERT in CML CD34+/blast cells, and the consequent telomere shortening with each division, is the direct cause of the shortened telomeres frequently observed in CML patients. These data challenge the widely accepted view that hTERT expression is always increased in tumorigenesis. It is possible that the Bcr-Abl fusion product may play a role in the down-regulation of hTERT observed in the HSC of CML patients in this study.

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia.

The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors--2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.

Modulation of natural killer and antibody-dependent cellular cytotoxicity by interferon and interleukin-2 in chronic myeloid leukemia patients in remission.

Our earlier studies demonstrated that about 55% of chronic myeloid leukemia (CML) patients in remission exhibited impaired natural killer (NK) cytotoxicity (low NK responders) while antibody-dependent cellular cytotoxicity (ADCC) of these patients, on chicken red blood cells as targets, was within normal range. In this paper, we have attempted to modulate the NK cytotoxicity of CML patients in remission with interferon (IFN) and interleukin-2 (IL-2) singly or together. ADCC using K562 target-directed monoclonal antibody (MAb) 4.6E10 was also modulated by treating the effectors with IFN or IL-2. Pretreatment of nonadherent mononuclear cells from peripheral blood (NAPBMNC) with IFN or IL-2 was found to result in 20 and 21% increase in target cell lysis in case of healthy donors, 79 and 98% increase in case of CML normal NK responders, and 164 and 159% increase in case of CML low NK responders. Combined use of IFN and IL-2 potentiated further the lymphocytotoxicity to 25% in healthy donors, 135% in normal NK responder CML patients and 283% in low NK responder CML patients. This treatment resulted in restoration of cytotoxicity of the latter group of patients to a normal level. The augmentation was seen in 80-100% CML patients. Although ADCC with chicken red blood cells as targets was within normal range, ADCC mediated with MAb to K562 cells was significantly lower in CML low NK responders (24.5%) than CML normal NK responders (42.4%) and healthy donors (65.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

PMN cells from chronic myeloid leukemia (CML) patients show defective chemotaxis in remission

The chemotactic index (C.I.) of granulocytes from chronic myeloid leukemia (CML) patients at diagnosis and in subsequent remission was measured using different concentrations of the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), by time lapse cinematography and compared with that of normal granulocytes. The C.I. of CML polymorphonuclear leucocytes (PMNL) at diagnosis and in remission was significantly lower than the C.I. of PMNL from normal subjects ( p < 0.001 and 0.05 > p > 0.02, respectively). PMNL from CML patients at diagnosis showed increased speed after stimulation with FMLP. In most of the CML patients, the highest values of C.I., speed and the number of motile cells were obtained at FMLP concentrations of 10–100-fold higher than those required for normal PMNL. These results suggest an alteration in the interaction between FMLP and its receptors and that events occurring after FMLP binding are also altered. It was earlier shown that PMNL from CML patients in the active stages of the disease show defective chemotaxis. Present studies show persistence of such defective cells in the peripheral blood of CML patients in remission. These results also suggest that defects in PMNL from CML patients may be constitutional.

Natural and antibody-dependent cellular cytotoxicity in chronic myeloid leukemia patients in remission

Non-adherent peripheral blood mononuclear cells (NA-PBMNC) from 67 chronic myeloid leukemia (CML) patients in the first and two subsequent remissions, and 23 normal healthy donors were tested for NK and ADCC activities in short term chromium release assays using K562 and antibody-coated chicken RBCs as respective targets. CML patients in remission exhibited significantly reduced NK cytotoxicity (16.1–19.7%) compared to normal healthy donors (47.4%). Of the patients tested, 55% exhibited NK levels below the mean percent cytotoxicity — 2SD (12.5%) of normal donors (low responders), while 45% exhibited NK cytotoxicity above the 12.5% level (normal responders). On the other hand, CML patients in remission showed ADCC activity comparable to that of normal healthy donors (53.3%) irrespective of whether they belonged to normal NK responder group (55.5–65.0% ADCC) or low NK responder group (39.4–48.3% ADCC). The low or normal NK responder status of CML patients was not found to be related to either progression on the disease, or the type of drug used to bring about remission, or to the period in remission at the time of testing. In-vitro treatment of effector lymphocytes with recombinant human IFN α resulted in augmented of NK activity in both low and normal NK responder patients. The IFN-augmented NK activity in low responder patients however remained below the normal levels.

Circulating immune complexes in chronic myeloid leukemia: Turbidimetric measurement and two-dimensional electrophoretic analysis

A 3.75% polyethylene glycol -6000 mediated turbidimetric method was used for the estimation of levels of circulating immune complexes (CIC) in sera of normal human subjects and patients with chronic myeloid leukemia (CML). In terms of equivalents of heat-aggregated gamma globulin (eq.HAGG mg/ml serum), the mean levels of CIC were: 5.69 mg/ml (SD 4.78) in 81 normal human subjects; 22.63 mg/ml (SD 9.86) in 52 untreated CML patients; 7.61 mg/ml (SD 5.74) in 54 CML patients in remission; and 21.70 mg/ml (SD 8.15) in 18 CML patients in relapse. High CIC levels, thus, showed a significant association with the pretreatment — and relapse — status of the disease ( p<0.001 and p<0.001, respectively). Though the levels decreased during remission, they were still significantly above the mean level in the controls ( p<0.05). The CIC level-disease status relationship was clearly evident in the serial studies on 19 CML patients who donated serum samples prior to treatment as well as during remission. Two-dimensional electrophoretic analysis of the CIC samples revealed the presence of a polypeptide (mol. wt approx. 37,000 and pI approx. 5.8) in 10 out of 18 CIC samples from the untreated CML patients. Such a moiety was not detected in six CIC samples from normal subjects. The association of this polypeptide with CML gains support from the observation that in 5 CML patients, this moiety was present in the CIC samples obtained prior to treatment but absent in the samples subsequently obtained during remission.

Agglutination of granulocytes from chronic myeloid leukaemia by concanavalin A.

Peripheral blood leucocytes from chronic myeloid leukaemia (CML) patients previously untreated or in relapse are agglutinated by concanavalin A (Con A) at concentrations of 25-400 microgram/ml. Leucocytes from normal subjects, from CML patients in remission and from patients with granulocytosis are not agglutinated by Con A. Normal bone marrow cells which contain immature and mature myeloid cells are not agglutinated either by Con A. These results suggest that an alteration occurs in the cell surface of myeloid cells in CML due to leukaemic change and results in the agglutination of these cells by Con A. If peripheral blood leucocytes of CML patients in relapse are separated into immature and mature fractions, both these fractions are agglutinated by Con A suggesting that this alteration occurs in the cell surface of both the immature and the mature myeloid cells.

Cellular sensitization in chronic myeloid leukaemia patients to leukaemic blast antigens

Sixteen chronic myeloid leukaemia (CML) patients in remission were tested with solubilized membrane antigens from CML leukaemic cells, CML blasts, AML blasts and ALL blasts for cellular immunity in vitro by lymphocyte transformation (LT) and leucocyte migration inhibition (LMI) assays. Twelve CML patients in remission were tested with allogeneic PHA-transformed normal lymphoblasts. As controls, peripheral-blood leucocytes from 9 healthy persons were tested with the same antigen preparations. It was seen that 8/16 (50%) CML patients responded to CML antigens by both LT and LMI assays, while 5/16 (31%) patients reacted to CML blasts and 44% (7/16) patients reacted to AML blast antigens. It was interesting to note that 5/11 (45%) CML patients reacted to ALL blast antigens by both assays. One out of 12 patients reacted to PHA-transformed lymphoblasts. None of the healthy controls reacted to leukaemia-associated antigens. The results suggest the sharing of antigens between myeloid leukaemic cells, myeloid blasts and lymphoid blasts.