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

AbstractTyrosine 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.

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BMS-214662 potently induces apoptosis of chronic myeloid leukemia stem and progenitor cells and synergizes with tyrosine kinase inhibitors

Blood ◽

10.1182/blood-2007-09-112573 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2843-2853 ◽

Cited By ~ 82

Author(s):

Mhairi Copland ◽

Francesca Pellicano ◽

Linda Richmond ◽

Elaine K. Allan ◽

Ashley Hamilton ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Blast Crisis ◽

Chronic Phase ◽

Hematopoietic Stem

Chronic myeloid leukemia (CML), a hematopoietic stem-cell disorder, cannot be eradicated by conventional chemotherapy or the tyrosine kinase inhibitor imatinib mesylate (IM). To target CML stem/progenitor cells, we investigated BMS-214662, a cytotoxic farnesyltransferase inhibitor, previously reported to kill nonproliferating tumor cells. IM or dasatinib alone reversibly arrested proliferation of CML stem/progenitor cells without inducing apoptosis. In contrast, BMS-214662, alone or in combination with IM or dasatinib, potently induced apoptosis of both proliferating and quiescent CML stem/progenitor cells with less than 1% recovery of Philadelphia-positive long-term culture-initiating cells. Normal stem/progenitor cells were relatively spared by BMS-214662, suggesting selectivity for leukemic stem/progenitor cells. The ability to induce selective apoptosis of leukemic stem/progenitor cells was unique to BMS-214662 and not seen with a structurally similar agent BMS-225975. BMS-214662 was cytotoxic against CML blast crisis stem/progenitor cells, particularly in combination with a tyrosine kinase inhibitor and equally effective in cell lines harboring wild-type vs mutant BCR-ABL, including the T315I mutation. This is the first report of an agent with activity in resistant and blast crisis CML that selectively kills CML stem/progenitor cells through apoptosis and offers potential for eradication of chronic phase CML.

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miRNome profiling of LSC-enriched CD34+CD38−CD26+ fraction in Ph+ CML-CP samples from Argentinean patients: a potential new pharmacogenomic tool

Frontiers in Pharmacology ◽

10.3389/fphar.2020.612573 ◽

2021 ◽

Vol 11 ◽

Author(s):

María Sol Ruiz ◽

María Belén Sánchez ◽

Simone Bonecker ◽

Carolina Furtado ◽

Daniel Koile ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Kinase Inhibitors ◽

Residual Disease ◽

Chronic Phase ◽

Term Therapy ◽

Hematopoietic Stem ◽

The Road ◽

Functional Studies ◽

Myeloid Progenitor Cells

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of LSC-enriched CD34+CD38−CD26+ and normal hematopoietic stem cells (HSC) fractions obtained from the same chronic phase (CP) CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC-enriched CD34+CD38−CD26+ and HSC fractions from CML-CP patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML-CP patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. We confirmed by RT-qPCR that the levels of miR-196a-5p were increased more than nine-fold in CD26+ (BCR-ABL1+) vs. CD26− (BCR-ABL1−) CD34+CD38− fractions from CML-CP patients at diagnosis, and in silico analysis revealed a significant association to lipid metabolism and hematopoiesis functions. In the light of recent descriptions of increased oxidative metabolism in CML LSC-enriched fractions, these results serve as a guide for future functional studies that evaluate the role of microRNAs in this process. Metabolic vulnerabilities in LSCs open the road for new therapeutic strategies. This is the first report of the miRNome of CML-CP CD34+CD38− fractions that distinguishes between CD26+ (BCR-ABL1+) and their CD26− (BCR-ABL1-) counterparts, providing valuable data for future studies.

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Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Nature Communications ◽

10.1038/s41467-020-20259-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hu Lei ◽

Han-Zhang Xu ◽

Hui-Zhuang Shan ◽

Meng Liu ◽

Ying Lu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Chronic Myelogenous Leukemia ◽

Drug Targets ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Myelogenous Leukemia ◽

Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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Determinants of Choice of Front-Line Tyrosine Kinase Inhibitor for Chronic Phase CML: A Study from the "Registro Italiano LMC & Campus CML"

Blood ◽

10.1182/blood-2020-135972 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-36

Author(s):

Mario Tiribelli ◽

Roberto Latagliata ◽

Massimo Breccia ◽

Isabella Capodanno ◽

Maria Cristina Miggiano ◽

...

Keyword(s):

High Risk ◽

Tyrosine Kinase ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Multivariable Analysis ◽

Chronic Phase ◽

Front Line ◽

Line Therapy ◽

Concomitant Medications

Introduction : therapy of chronic phase (CP) chronic myeloid leukemia (CML) is based on tyrosine kinase inhibitors (TKIs) in virtually all patients. Three TKIs are approved for first-line therapy in Italy: imatinib and two second-generation (2G) TKIs, dasatinib and nilotinib. Choice of the front-line TKI is based on a combined evaluation of patient's and disease characteristics, age, risk, comorbidities and concomitant medications. Treating physician's preference and, in some cases, economic considerations, particularly after the advent of generic imatinib, may play a role in TKI selection. However, to date, few data are available on TKI use in a whole nation and on the possible drivers of treatment choice. Aim of the present work was to analyse the use of front-line TKI therapy in a large, unselected cohort of Italian CP-CML patients, correlating patient's features to drug choice. Methods: in the framework of the national Campus CML program, we retrospectively evaluated 1422 patients with CP-CML diagnosed from 2012 and 2019 in 21 haematologic Centres, mostly in academic and/or tertiary hospitals, widespread through the entire Italian territory and treated frontline with imatinib, dasatinib or nilotinib. Results: median age at diagnosis was 59.9 years [interquartile range (IQR) 47.1 - 71.7], with 317 (22.3%) patients under 45 years, 552 (38.8%) between 45 and 65 years and 553 (38.9%) older than 65 years; 821 (57.7%) patients were males. Among 1364 evaluable patients, CML risk according to Sokal score was low in 540 (39.6%), intermediate in 610 (44.7%) and high in 214 (15.7%) patients respectively; the number at low, intermediate or high risk according to the novel ELTS score among 1325 evaluable patients was 759 (57.3%), 402 (30.3%) and 164 (12.4%) respectively. Considering comorbidities, 1003 (70.6%) patients had at least one active disease at the time of CML diagnosis, the most common being hypertension (n=547, 38.5%), previous neoplasms (n=185, 13.0%), diabetes (n=150, 10.6%), chronic bronchopulmonary diseases (n=114, 8.0%), acute myocardial infarction (n=95, 6.7%), previous stroke (n=36, 2.5%) and other vascular diseases (n=98, 6.9%). Among 1335 evaluable patients, 813 (60.9%) were taking at least one concomitant medication, with 280 (21.0%) taking 3-5 drugs and 140 (10.5%) taking 6+ drugs at time of TKI start. As to the frontline therapy, 794 (55.8%) received imatinib and 628 (44.2%) were treated with 2G-TKIs, (226 dasatinib and 402 nilotinib) respectively. According to age, 2G-TKIs were chosen for majority of patients aged <45 (69.1%) while imatinib was used in 76.9% of patients over 65 (p<0.001). There was a predominance of imatinib use across all Sokal (51.1% in low, 61.3% in intermediate and 51.4% in high) and ELTS (50.3% in low, 60.4% in intermediate and 66.5%) risk categories. We observed a prevalent use of 2G-TKIs in patients presenting with higher WBC counts (55.1% if WBC >100,000/mm3 vs 38.2% if WBC <100,000/mm3; p<0.001), lower Hb (53.8% if Hb <10 g/dl vs 41.9 if Hb >10 g/dl; p=0.001) and bigger spleen (65.1% if spleen >5 cm vs 44.8% if spleen 1-5 cm vs 37.3% if spleen not palpable; p<0.001). There was a decreasing use of 2G-TKIs with higher number of concomitant drugs: 64.4% for 0, 47.7% for 1-2, 27.0% for 3-5 and 13.6% for >5 drugs, respectively (p<0.001). Concordantly, there was a significant higher use of imatinib in patients with hypertension (69.8%), diabetes (70.0%), COPD (73.7%), previous neoplasms (73.0%), AMI (86.3%) or stroke (97.2%) history (p<0.001 for all conditions). Lastly, we observed a wider use of imatinib (61.1%) in patients diagnosed in years 2018-19, compared to those of the period 2012-17 (53.7%; p=0.01). In multivariable analysis, factors correlated with imatinib use were age > 45 years, intermediate or high Sokal risk, presence of some comorbidities (2nd neoplasia and stroke) and number of concomitant medications. Conclusions: preliminary results of this observational study on almost 1500 patients show that around 55% of newly diagnosed Italian CP-CML patients receive imatinib as front-line therapy, and that the use of 2G-TKI is prevalent in the younger patients and in those with no concomitant clinical conditions. The counterintuitive finding of imatinib prevalence as frontline treatment in high risk patients might be explained by the older age of these patients. Introduction of the generic formulation in 2018 seems to have fostered the use of imatinib. Figure Disclosures Breccia: Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Abbvie: Consultancy; Bristol-Myers Squibb/Celgene: Consultancy, Honoraria. Cavazzini:Pfize: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Saglio:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Ariad: Research Funding; Roche: Research Funding.

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Nilotinib therapy in an imatinib intolerant chronic myeloid leukemia patient with pulmonary severe hypertension

Clinical Management Issues ◽

10.7175/cmi.v4i2s.1072 ◽

2015 ◽

Vol 4 (2S) ◽

pp. 17-20

Author(s):

Mario Annunziata

Keyword(s):

Side Effects ◽

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Chronic Phase ◽

Chronic Myeloid Leukemia Patient ◽

Cytogenetic Response ◽

Myelogenous Leukemia ◽

Molecular Response ◽

Cutaneous Rash

Imatinib mesylate is a tyrosine kinase inhibitor that has significant efficacy in the treatment of chronic myelogenous leukemia. In general, hematologic and extrahematologic side effects of imatinib therapy are mild to moderate, with the large majority of patients tolerating prolonged periods of therapy. However, a minority of patients are completely intolerant of therapy, while others are able to remain on therapy despite significant side effects. Here, we describe a chronic phase CML patient with pulmonary arterial hypertension, mechanical hearth valve, who experienced extrahematologic adverse event (persistent grade III cutaneous rash, despite two discontinuations of imatinib and using of steroid). Necessitating switch to one of new tyrosine kinase inhibitors, nilotinib, has resulted in complete cytogenetic response and major molecular response, after 3 and 6 months, respectively. No cross-intolerance with imatinib was observed during nilotinib therapy. Besides, this clinical case suggests that warfarin and nilotinib can be used concurrently without the risk of increased anticoagulant effect.

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Targeting Mcl-1 Enhances the Activity of Tyrosine Kinase Inhibitor Gilteritinib in FLT3 Mutated AML

Blood ◽

10.1182/blood-2020-141399 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Vivian Ruvolo ◽

Wenjing Tao ◽

Paul Hughes ◽

...

Keyword(s):

Tyrosine Kinase ◽

Signaling Pathways ◽

Progenitor Cells ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Mapk Signaling ◽

Culture Conditions ◽

Beta Catenin ◽

Adaptive Resistance

Anti-apoptotic Bcl-2 proteins play critical roles in AML cell and AML stem/progenitor cell survival and drug resistance, hence are relevant therapeutic targets. Indeed, the combination of the selective Bcl-2 inhibitor venetoclax (VEN) with a hypomethylating agent elicits CR/CRi rates of > 65%, is well tolerated by elderly AML patients, and obtained FDA approval. However, despite of the major improvement in response rates, survival extension was limited and most patients ultimately relapsed largely due to the development of resistant disease. Molecular analysis of treated patients revealed that primary and adaptive resistance to VEN-based combinations was frequently characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS (DiNardo CD et al., Blood 2020). FLT3 is one of the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways such as RAS/MAPK, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients treated with TKIs ultimately relapse and adapt to TKI therapy by reactivating the MAPK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 expression can be induced by VEN treatment and is a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML, targeting AML cells and stem/progenitor cells. To determine if targeting Mcl-1 enhances the activity of TKIs in FLT3 mutated AML, we treated MV4-11 and Molm13 cells with Mcl-1 inhibitor AMG176 and TKI gilteritinib (GIL) and observed synergism, as defined by combination index < 1 in both cells. Mechanistic studies demonstrated that GIL markedly decreased Mcl-1 and antagonized AMG176-induced Mcl-1 induction. GIL and its combination with AMG176 also decreased Bcl-xL. Although Bcl-2 protein levels were largely not changed in MV4-11 cells, we found both single treatment and the combination greatly decreased Bcl-2 associated athanogene (BAG) proteins BAG1, BAG3, and BAG4 at the RNA level, which needs to be confirmed at the protein level. The BAG proteins are a family of chaperone regulators and BAG1 was reported to bind and enhance the activity of multiple proteins known to support cells survival, including Bcl-2 (Takayama S et al., Cell 1995). Interestingly, GIL treatment greatly diminished the levels of beta-catenin and its target protein c-Myc, consistent with our previous report that FLT3 regulates beta-catenin signaling (Xiang et al., CCR, 2018). We have generated Mcl-1 overexpressing (OE) and VEN-resistance (VEN-R) MV4-11 and Molm13 cells. The Mcl-1 OE cells are highly resistant to VEN and the VEN-R cells expressed high levels of Mcl-1. Combined inhibition of AMG176 and GIL synergistically induced cell death in Mcl-1 OE and VEN-R resistant cells. Although the expression is low in AML cells we tested, BCL2A1 is also known as a resistant factor to VEN. We generated BCL2A1 OE MV4-11 and Molm13 cells and demonstrated that combined inhibition of FLT3 and Mcl-1 was highly effective in these cells as well. Western blot analysis revealed that GIL effectively decreased Mcl-1 in Mcl-1 OE and VEN-R and BCL2A1 in BCL2A1 OE MV4-11 cells. Next, we treated FLT3 mutated AML patient samples harboring both, ITD and D835 mutations, from 2 patients who had both failed VEN-based therapy and from 1 patient with ITD mutation, with AMG176 and GIL under MSC co-culture conditions. Synergy was observed in all samples in AML blasts and AML stem/progenitor cells. Collectively, our data demonstrate that targeting Mcl-1 enhances the activity of GIL in FLT3 mutated AML, including those resistant to/relapsed from VEN-based therapy, findings that may warrant clinical evaluation. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding. Hughes:Amgen: Current Employment. Chen:Amgen: Current Employment. Morrow:Amgen: Current Employment. Andreeff:Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

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A Retrospective Analysis about Frequency of Monitoring in Italian Chronic Myeloid Leukemia Patients after Discontinuation

Journal of Clinical Medicine ◽

10.3390/jcm9113692 ◽

2020 ◽

Vol 9 (11) ◽

pp. 3692

Author(s):

Matteo Dragani ◽

Giovanna Rege Cambrin ◽

Paola Berchialla ◽

Irene Dogliotti ◽

Gianantonio Rosti ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Chronic Phase ◽

Molecular Response ◽

Molecular Monitoring ◽

Delay In Diagnosis ◽

Number Of Patients

Successful discontinuation of tyrosine kinase inhibitors has been achieved in patients with chronic-phase chronic myeloid leukemia (CML). Careful molecular monitoring after discontinuation warrants safe and prompt resumption of therapy. We retrospectively evaluated how molecular monitoring has been conducted in Italy in a cohort of patients who discontinued tyrosine kinase inhibitor (TKI) treatment per clinical practice. The outcome of these patients has recently been reported—281 chronic-phase CML patients were included in this subanalysis. Median follow-up since discontinuation was 2 years. Overall, 2203 analyses were performed, 17.9% in the first three months and 38.4% in the first six months. Eighty-six patients lost major molecular response (MMR) in a mean time of 5.7 months—65 pts (75.6%) during the first six months. We evaluated the number of patients who would experience a delay in diagnosis of MMR loss if a three-month monitoring schedule was adopted. In the first 6 months, 19 pts (29.2%) would have a one-month delay, 26 (40%) a 2-month delay. Very few patients would experience a delay in the following months. A less intense frequency of monitoring, particularly after the first 6 months off treatment, would not have affected the success of treatment-free remission (TFR) nor put patients at risk of progression.

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Recurrent bone marrow aplasia secondary to nilotinib in a patient with chronic myeloid leukemia: A case report

Journal of Oncology Pharmacy Practice ◽

10.1177/1078155212438112 ◽

2012 ◽

Vol 18 (4) ◽

pp. 440-444 ◽

Cited By ~ 6

Author(s):

Prathima Prodduturi ◽

Anamarija M Perry ◽

Patricia Aoun ◽

Dennis D Weisenburger ◽

Mojtaba Akhtari

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Chronic Phase ◽

Bone Marrow Aplasia ◽

Line Therapy ◽

Grade 3

Nilotinib is a potent tyrosine kinase inhibitor of breakpoint cluster region-abelson (BCR-ABL), which has been approved as front-line therapy for newly diagnosed chronic myeloid leukemia in chronic phase and as second-line therapy after imatinib failure in chronic or accelerated phase chronic myeloid leukemia. Tyrosine kinase inhibitors have been associated with myelosuppression and grade 3 or grade 4 cytopenias are not uncommon in chronic myeloid leukemia patients treated with these drugs. There are a few reports of imatinib-associated bone marrow aplasia, but to our knowledge only one reported case of bone marrow aplasia associated with nilotinib. Herein, we report a 49-year-old male patient with chronic phase chronic myeloid leukemia, who developed severe bone marrow aplasia due to nilotinib. Possible mechanisms for this significant adverse drug reaction are discussed along with a review of literature.

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Protection of CML Progenitors from Bcr-Abl Tyrosine Kinase Inhibitor Mediated Apoptosis by the Bone Marrow Stromal Microenvironment.

Blood ◽

10.1182/blood.v110.11.3378.3378 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3378-3378

Author(s):

Bin Zhang ◽

Heiko Konig ◽

Tinisha Mcdonald ◽

Tessa L. Holyoake ◽

Dario Campana ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Direct Contact ◽

Hematopoietic Stem ◽

Transwell Insert ◽

Abl Tyrosine Kinase ◽

Stromal Microenvironment ◽

Cd34 Cells ◽

Tki Treatment

Abstract The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant hematopoietic stem and progenitor cells (HSPC). The bone marrow microenvironment regulates the self-renewal, proliferation and differentiation of HSPC. We investigated the role of microenvironmental interactions in resistance of CML HSPC to elimination by BCR-ABL tyrosine kinase inhibitors (TKI). CML CD34+CD38− primitive progenitor cells and CD34+CD38+ committed progenitor cells were cultured for 96 hours with IM (5μM), nilotinib (5μM) and dasatinib(150nM), in medium supplemented with low concentrations of growth factors, with and without irradiated primary human marrow stromal cells (immortalized by ectopic telomerase expression) followed by an assessment of apoptosis and proliferation. Culture with stroma did not result in significant alteration of apoptosis in the absence of TKI treatment (3.1±0.7% apoptosis for primitive progenitors with stroma and 2.7±0.9% without stroma, 3.7±0.2% for committed progenitors with stroma and 4.7±2.1% without stroma). Coculture with stroma completely protected CML primitive and committed progenitors from TKI-induced apoptosis. CML CD34+CD38− cells demonstrated 20±6% apoptosis following culture with IM in the absence of stroma, but only 3.8±1% apoptosis in the presence of stroma (p=0.04, n=4). Similarly, apoptosis with nilotinib decreased from 12.5±1.8% without stroma to 2.9±0.3% with stroma (p=0.033), and apoptosis with dasatinib decreased from 7.1±0.04% without stroma to 2.7±0.2% with stroma (p=0.001). Apoptosis of CML CD34+CD38+ cells also significantly decreased following TKI treatment with 12.9±4.0%, 10.6±3.2%, 8.4±2.3% apoptosis observed after IM, nilotinib and dasatinib treatment respectively without stroma and 7.1±1.2%, 4.8±1.0%, 3.7±0.4% with stroma, (p=0.04, p=0.03 and p=0.02 respectively, n=4). Culture with stroma resulted in mild reduction in CML progenitor proliferation in the absence of TKI treatment, but TKI treatment resulted in similar degrees of inhibition of proliferation regardless of the presence of stroma. Culture of CML CD34+ cells in a Transwell insert with 0.45μm pores, allowing free diffusion of stromal factors but preventing direct contact with stroma, was associated with reduction in the protective effect of stroma coculture (32.2% apoptosis without stroma, 14.7% with stroma, and 24.6% with Transwell insert). Addition of blocking antibodies to a4 integrin and N-cadherin did not affect survival of CML CD34+ cells in the absence of IM, but resulted in enhanced apoptosis of CML CD34+ cells cocultured with stroma after addition of IM (20.4% apoptosis without antibody, 28.9% with anti-N-cadherin, and 29.8% with anti-integrin antibody). We conclude that the bone marrow stromal microenvironment protects CML primitive and committed progenitors from pro-apoptotic effects of BCR-ABL TKI treatment. Direct contact-mediated interactions, likely through VLA-4 and N-Cadherin, play an important role in protecting CML CD34+ cells from TKI-mediated apoptosis. These observations indicate that measures aimed at interfering with the protective effects of stroma could be of benefit for the eradication of residual malignant progenitors in CML patients receiving BCR-ABL TKI treatment.

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Inhibition of Bcl-2/Bcl-XL Promotes Apoptosis in Blast Crisis CML Including Quiescent Primitive Progenitor Cells Regardless of Cellular Responses to Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v114.22.646.646 ◽

2009 ◽

Vol 114 (22) ◽

pp. 646-646

Author(s):

Duncan H. Mak ◽

Wendy D. Schober ◽

Marina Konopleva ◽

Jorge Cortes ◽

Hagop M. Kantarjian ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Progenitor Cells ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Blast Crisis ◽

Antiapoptotic Proteins ◽

Cell Compartments ◽

Cd34 Cells

Abstract Abstract 646 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor revolutionized the treatment for patients with CML. Development of resistance, limited activity in blast crisis CML, and more importantly, insensitivity of quiescent primitive CD34+ CML progenitor cells are evolving problems facing this therapy. Antiapoptotic Bcl-2 proteins were known to be highly expressed in Bcr-Abl expressing cells and inhibition of Bcl-2/Bcl-XL by the selective inhibitor ABT-737 was reported to augment the killing of tyrosine kinase inhibitors in CML cells. However, its effect on quiescent primitive CD34+ CML progenitor cells is unknown. To investigate the effect of activating the apoptotic machinery in quiescent primitive CD34+CML progenitor cells, which are resistant to current therapies, we first compared the expression of antiapoptotic proteins in proliferating and quiescent primitive CD34+CML progenitor cells. Cells obtained from patients with blast crisis CML were stained with the fluorescent 5-(and 6-) carboxy-fluorescein diacetate succinimidyl ester, a cell proliferation tracking dye, and cultured in vitro for 4-6 days. Cells were then stained with CD34 antibody and FACS sorted into proliferating and quiescent CD34+/PI- CML progenitor cells. RNA levels of antiapoptotic proteins in these two cell populations (n=8) were determined by real-time RT-PCR: quiescent and proliferating primitive CD34+ CML progenitor cells expressed similar levels of Bcl-2, Bcl-XL, Mcl-1, and XIAP implying that like total blast cells, quiescent primitive CD34+CML progenitor cells may also be sensitive to agents targeting these proteins. We next treated 5 samples obtained from patients with blast crisis CML with ABT-737 and measured apoptosis in total CD34+ cells, proliferating CD34+ cells, and quiescent CD34+ cells. All 5 patients were resistant to or relapsed from imatinib and nilotinib and/or dasatinib treatments and they were insensitive to imatinib in vitro as expected. However, cells from 4 patients were sensitive to ABT-737, in bulk blasts and in both proliferating and quiescent CD34+ CML cell compartments: % specific apoptosis with 100 nM of ABT-737=40.8±7.7, 38.4±8.5, 40.0±5.1, respectively at 24 hours. Interestingly, when ABT-737 was combined with imatinib, cell death was greatly enhanced in cells from all 5 patients in all cell compartments (combination index=0.059±0.032, 0.041±0.025, 0.111±0.042, respectively). Furthermore, we showed previously, that triptolide, an antitumor agent from a Chinese herb, induces apoptosis in both proliferating and quiescent primitive CD34+CML progenitor cells by decreasing Mcl-1 which is a resistant factor for ABT-737, XIAP, and Bcr-Abl protein levels (Mak D. et al., MCT in press). When ABT-737 was combined with triptolide, a significant increase of cell death was found in total CD34+ and proliferating as well as quiescent primitive CD34+CML cells with combination index at EC50=0.57, 0.55, and 0.56, respectively in cells from the 5 patients suggesting a high degree of synergism. In summary, Bcl-2, Bcl-XL, Mcl-1, and XIAP are equally expressed in proliferating and quiescent primitive CML cells and targeting Bcl-2/Bcl-XL promotes death of blast crisis CML cells, tyrosine kinase inhibitor resistant CML cells, and quiescent primitive CD34+ CML progenitor cells. Researches suggest that the combination of apoptosis inducing agents and tyrosine kinase inhibitor is a novel strategy to overcome tyrosine kinase resistance, eradicate quiescent primitive CML progenitor cells, and improve current therapy for patients with CML. Disclosures: No relevant conflicts of interest to declare.

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