Chronic Myeloid Leukemia Stem Research Articles

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach

BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.

Characterizing Population Heterogeneity and Signaling Changes in Chronic Myeloid Leukemia Stem and Progenitor Cells upon Combined Treatment with Imatinib and MEK Inhibitors Using Quantitative Single Cell Phospho-Imaging

Chronic myeloid leukemia (CML) is caused by the oncogenic tyrosine kinase BCR-ABL1, which is uniquely present in the leukemic cells and drives progression of the disease. Small-molecule ABL1 tyrosine kinase inhibitors (TKIs) have transformed prognosis for patients as compared to interferon or standard chemotherapy, with the vast majority of patients achieving deep, long-term remission on treatment (Druker et al., Nature Medicine 2009). However, results from multiple TKI discontinuation studies over the past decade suggest approximately 50-60% of patients must remain on therapy indefinitely, highlighting challenges associated with the retention of a residual pool of BCR-ABL1-positive leukemic stem cells (LSCs) (Mahon et al., The Lancet 2018; Ross et al., Blood 2013). Previously we have shown that, while ABL1 TKIs can effectively inhibit BCR-ABL1 kinase activity and reduce CML disease burden, LSCs avoid ABL1 TKI-induced cell death via alternate signaling pathways such as MEK/ERK and are able to persist (Corbin et al., JCI 2011). Detecting changes in phospho-activation of such alternative pathways at the single cell level using primary CML samples has yet to be characterized and will inform therapeutic options for complete disease eradication. Here, we dissect phospho-signaling diversity with single cell level granularity across heterogeneous subpopulations of CML stem (CD34+CD38-) and progenitor (CD34+CD38+) cells upon drug treatment using a new quantitative imaging method that allows for sensitive detection of critical changes in signaling (Jacob et al., Scientific Reports 2016). Briefly, primary CD34+ cells were isolated from newly diagnosed TKI naïve CML patients by MACS column and cultured ex-vivo in the presence of imatinib and MEK inhibitors (trametinib or cobimetinib), either alone or in combination. Cells were then assessed for apoptosis by flow cytometry annexin assay, followed by fixing/staining for CD38, pCRKL, and pERK, and quantified for changes in signaling upon treatment by quantitative imaging method. This new phospho-imaging method allows for low cell number input and maximizes data output per sample, opening opportunities to assess translational applicability of combinations in a highly biologically relevant context of small but critical LSC populations. Quantification of phospho-signal revealed that pCRKL was reduced in a dose dependent manner across a range of concentrations of imatinib in both CML stem and progenitor cells. In contrast, while pERK levels were also reduced with imatinib treatment, a subset of CML stem cells exhibited high levels of pERK signaling even at high (5uM) concentrations of imatinib. Combination treatment with imatinib and trametinib or cobimetinib markedly increased apoptosis in CML CD34+ cells compared to each single agent. Within the stem cell population, median pERK fluorescence was significantly reduced with imatinib or trametinib compared to untreated control (p<0.0001), and further reduced with the combination compared to each single agent (p<0.0001 and p=0.04, respectively). Furthermore, the percent of cells with pERK levels below the untreated median value was 87%, 90% and 97% upon treatment with imatinib, trametinib or the combination respectively. Together, our data suggest that variable levels of ERK phosphorylation exist within the CML stem and progenitor compartments, including a subset of CML stem cells that exhibit persistently high levels of pERK despite effective suppression of BCR-ABL1 kinase activity by imatinib. This stem cell persistence warrants the need for combination strategies to inhibit BCR-ABL1 and MEK/ERK for their complete elimination. DisclosuresTyner:AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Leap Oncology: Equity Ownership; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Agios: Research Funding. Druker:Celgene: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oregon Health & Science University: Patents & Royalties; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Novartis Pharmaceuticals: Research Funding; ARIAD: Research Funding; Millipore: Patents & Royalties; Amgen: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding.

Reactive oxygen species in BCR-ABL1-expressing cells - relevance to chronic myeloid leukemia.

Chronic myeloid leukemia (CML) results from the t(9;22) reciprocal chromosomal translocation producing the BCR-ABL1 gene, conferring growth and proliferation advantages in the CML cells. CML progresses from chronic, often syndrome-free, to blast phase, fatal if not treated. Although the involvement of BCR-ABL1 in some signaling pathways is considered as the cause of CML, the mechanisms resulting in its progression are not completely known. However, BCR-ABL1 stimulates the production of reactive oxygen species (ROS), which levels increase with CML progression and induce BCR-ABL1 self-mutagenesis. Introducing imatinib and other tyrosine kinase inhibitors (TKIs) to CML therapy radically improved its outcome, but TKIs-resistance became an emerging problem. TKI resistance can be associated with even higher ROS production than in TKI-sensitive cells. Therefore, ROS-induced self-mutagenesis of BCR-ABL1 can be crucial for CML progression and TKI resistance and in this way should be taken into account in therapeutic strategies. As a continuous production of ROS by BCR-ABL1 would lead to its self-destruction and death of CML cells, there must be mechanisms controlling this phenomenon. These can be dependent on DNA repair, which is modulated by BCR-ABL1 and can be different in CML stem and progenitor cells. Altogether, the mechanisms of the involvement of BCR-ABL1 in ROS signaling can be engaged in CML progression and TKI-resistance and warrant further study.

Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia.

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34+ CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34+ CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34+ CP-CML cell engraftment in NSG mice and, upon administration to EGFP+ /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Design, Optimization, and Pre-Clinical Evaluation of Direct, Mechanism-Based STAT3 Inhibitors for Treating Myeloid Disorders

We have identified STAT3 as a convergence point for oncogenic signaling in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) lacking BCR-ABL1 kinase domain mutations. In addition, we found that STAT3 activity contributes to disease in other myeloid disorders, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Utilizing TKI-resistant CML as a model system, we identified BP-5-087 as a small molecule inhibitor of STAT3 that reduces STAT3 phosphorylation and nuclear transactivation (Eiring et al. Leukemia, 2014).Binding of BP-5-087 to the STAT3 SH2 domain was initially assessed using fluorescence polarization (FP) assays and high-resolution computational docking simulations. To further validate the binding motif of BP-5-087, we conducted time-resolved electrospray ionization mass spectrometry/hydrogen-deuterium exchange experiments. Fold-change in deuterium uptake was analyzed for 68 STAT3 peptides representing 71% sequence coverage, and mapped onto the crystal structure of STAT3. This analysis precisely defined the binding epitope for BP-5-087 within the STAT3 SH2 domain.We next tested the effects of BP-5-087 in several myeloid malignancies using relevant disease models. (i) CML stem and progenitor cells from TKI-resistant patients without kinase domain mutations were treated with BP-5-087 ex vivo, using short-term liquid culture, clonogenic and LTC-IC assays. BP-5-087 treatment significantly reduced colony formation by CML stem and progenitor cells (p<0.01), with no effect on normal human CD34+ cord blood (CB) cells. (ii) Similarly, BP-5-087 also increased apoptosis and reduced viability (p<0.05) of primary AML blasts treated ex vivo with BP-5-087 for 72 hours in liquid culture. (iii) CD34+ cells from patients with myelofibrosis were also treated with BP-5-087 in clonogenic assays, and similar to CML, BP-5-087 reduced myeloid colony formation, although to a lesser extent.The in vivo activity of BP-5-087 was next evaluated in a murine model of JAK2 V617F-induced MPN. Briefly, Balb/c bone marrow was transduced with JAK2 V617F-GFP, followed by injection into lethally irradiated recipients. After disease induction, mice were treated with BP-5-087 (25 mg/kg) by once-daily oral gavage. No toxicities were observed after 40 days of treatment in BP-5-087-treated mice. While BP-5-087 did not significantly reduce the percentage of GFP+ cells, there was a 41% reduction of spleen weight in BP-5-087-treated mice compared to vehicle-treated controls (p<0.05). Post study analysis revealed BP-5-087 plasma concentrations <1 μM, suggesting that insufficient bioavailability contributed to the modest in vivo effects.To advance the lead optimization of our STAT3 inhibitor series, we instituted a comprehensive screening cascade. We first developed a computational model (quantitative structure-activity relationship, QSAR) to guide and prioritize selection of new inhibitor candidates for synthesis. Compounds are initially ranked using a methanethiosulfonate (MTS)-based cell viability assay in a TKI-resistant, STAT3-dependent CML cell line (AR230R). Inhibition of STAT3 is confirmed using a cell-based STAT3 reporter assay and an in vitro FP-based binding assay. Optimization of potency is balanced by the goals of reducing molecular weight (MW) and calculated LogP (cLogP) compared to BP-5-087 (MW: 694.8; cLogP: 7.3). Compounds with improvements in these categories are then subject to toxicity testing utilizing clonogenic assays with CD34+ CB cells. Non-toxic compounds are evaluated for their pharmacokinetic profile in Balb/c mice and tested for activity in primary samples from CML, AML and MPN patients. These activities have directed us to a lead compound, AM-1-124, which displays significant improvements in potency, MW, cLogP, and in vivo half-life compared to BP-5-087. AM-1-124 had minimal effects in the CB toxicity assay and induced apoptosis in primary AML patient samples at 2-fold lower concentrations than BP-5-087. With AM-1-124 as our current lead compound, we are continuing our iterative evaluation of novel STAT3 inhibitors utilizing our screening cascade. Design and testing of optimized, orally active inhibitors will enable further evaluation of STAT3 as a target in animal models of myeloid leukemia and will justify the clinical development of these compounds for patients in need of new targeted therapies. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

The Oncogenic Transcription Factor STAT5 Triggers Aberrant Expression Of CD25 (IL-2RA) In Neoplastic Stem Cells In Ph+ CML

Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL-targeting tyrosine kinase inhibitors (TKI), many patients relapse which may be due to resistance of neoplastic stem cells (NSC) against TKI. So far, little is known about specific markers and targets expressed on CML NSC. We here report that CML NSC express the interleukin-2R alpha-chain CD25 in a specific (aberrant) manner. Whereas normal bone marrow (BM) stem cells were found to express only low amounts or did not express CD25, CD34+/CD38− NSC in CML were found to express CD25 in almost all patients examined (31/33=95%), independent of the phase of disease. CML NSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), Campath-1 (CD52), KIT (CD117) and IL-3RA (CD123). We were also able to show that highly purified CD25+ NSC express BCR/ABL and engraft irradiated NOD-SCID-IL-2Rg-/- (NSG) mice with BCR/ABL+ cells, whereas CD34+/CD38-/CD25-/CD26- cells from the same patients failed to express BCR/ABL, and engrafted NSG mice with BCR/ABL-negative cells. To define signalling-molecules contributing to the expression of CD25 in CML NSC, we employed primary murine hematopoietic cells infected with bcr/abl-p210 in combination with a retrovirus encoding for STAT5A or STAT5B. Enforced expression of either STAT5A or STAT5B resulted in enhanced expression of CD25 in Lin-/Sca-1+/Kit+ mouse BM stem cells (LSK cells), regardless of the presence or absence of bcr/abl. In fact, the CD25-promoting effect of STAT5 was seen in normal stem cells as well as in bcr/abl-transformed LSK cells. Correspondingly, shRNA against STAT5 was found to downregulate expression of CD25 in the human CML cell line KU812. The BCR/ABL-targeting TKI imatinib, nilotinib and ponatinib were also found to inhibit expression of CD25 on KU812 cells. In primary CML NSC, ponatinib was found to induce marked suppression of CD25, whereas imatinib and nilotinib showed only weak effects. In consecutive experiments, we found that the MEK inhibitors RDEA119 and PD032509, the STAT5-targeting drug pimozide and the dual PI3 kinase/mTOR blocker BEZ235 all inhibit 3H-thymidine uptake and thus proliferation in KU812 cells. However, whereas RDEA119 and PD032509 as well as pimozide were found to downregulate expression of CD25 in KU812 cells, BEZ235 was found to even upregulate expression of CD25 on KU812 cells. We next applied an shRNA against CD25. The shRNA-induced knock-down of CD25 in KU812 resulted in an increased proliferative capacity, suggesting that CD25 may act as a potential tumor suppressor in CML stem- and progenitor cells. Together, our data show that CD25 is a novel functionally relevant marker-antigen and potential tumor suppressor of CML NSC. Expression of CD25 in CML cells appears to be triggered by MEK and STAT5 activity, and may be negatively regulated by the PI3 kinase/mTOR pathway. Whether modulation of CD25 can be exploited clinically and used as basis for the development of novel NSC-targeting treatment concepts in CML remains to be determined in future studies. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Genome-wide comparison of the transcriptomes of highly enriched normal and chronic myeloid leukemia stem and progenitor cell populations

The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.

Combined effects of the hedgehog pathway inhibitor LDE225 and nilotinib in a random mutagenesis screen

Recent studies have demonstrated that hedgehog pathway is activated in chronic myeloid leukemia (CML) stem cells via up-regulation of Smoothened (Smo), a seven transmembrane domain receptor protein. LDE225 is a small molecule Smo antagonist which has entered Phase I clinical evaluation in patients with solid tumors. We performed a comprehensive drug combination experiment using a broader range of concentrations for LDE225 and nilotinib. Compared with single agents, the combination of LDE225 and nilotinib was more effective at reducing the outgrowth of resistant cell clones. No outgrowth was observed in the presence of 2 μM nilotinib plus 20 μM LDE225. Also co-treatment with LDE225 and nilotinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to LDE225 and nilotinib even in BaF3 cells expressing T315I. To assess the in vivo efficacy of LDE225 and nilotinib, athymic nude mice were injected s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL mutation. 7 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, LDE225 (20 mg/kg; p.o. once every day), nilotinib (30 mg/kg; p.o. once every day), LDE225 (20 mg/kg; p.o. once every day) + nilotinib (30 mg/kg; p.o. once every day). The LDE225 and nilotinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or nilotinib- or LDE225-treated mice. Histopathologic analysis of tumor tissue from LDE225 plus nilotinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL staining. To investigate combined effects of LDE225 and nilotinib on primary Ph-positive acute lymphocytic leukemia (ALL) cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear cells from a Ph positive ALL patient. Treatment with LDE225 and nilotinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity and the endosteal surface. These results suggest that the combination with a Smo inhibitor and ABL TKIs may help to eliminate the Ph positive ALL cells. Taken together, the present study shows that the combination of LDE225 and nilotinib exhibits a desirable therapeutic index that can reduce the in vivo growth of mutant forms of BCR-ABL-expressing cells.