Primitive Chronic Myeloid Leukemia Cells Research Articles

Restoration of Mir-185 in Combination with BCR-ABL Downregulation By Therapeutic Delivery of siRNA with Lipid Nanoparticle Carriers Targets Drug Resistant Leukemic Stem/Progenitor Cells

Background: Despite development of effective tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), TKI therapy is not curative as some patients develop drug resistance or are at risk of disease relapse. Importantly, CML leukemic stem cells (LSCs) remain largely unresponsive and represent a key population for sustaining disease and relapse. We previously demonstrated that miR-185 levels were significantly reduced in CD34 + stem/progenitor cells from TKI-nonresponders compared to TKI responders. Further, restoration of miR-185 levels in CML LSCs and their progenitors impaired their survival and sensitized them to TKI in vitro and in vivo. However, the therapeutic potential of downregulating BCR-ABL using siRNA in combination with miR-185 mimic treatment to target CML LSCs have yet to be elucidated. Objective: Downregulation of BCR-ABL using siRNA represents a promising therapeutic approach to inhibit its kinase and non-kinase functions to combat drug-resistant CML and overcome limitations of current therapy. Additionally, previous work suggests that restoring miR-185 levels may improve therapeutic outcomes from complimentary therapies in CML, particularly in combination with BCR-ABL inhibition. Therefore, we aim to investigate the therapeutic outcomes and mechanism of action of inhibiting BCR-ABL using siRNA in combination with miR-185 mimic treatment in drug-resistant CML. Methods: To assess the effects of BCR-ABL downregulation in combination with miR-185 restoration, we encapsulated siRNA targeting BCR-ABL and a miR-185 mimic using an optimized lipid nanoparticle (LNP) formulation. Biological effects and mechanistic changes of this novel RNA combination was evaluated using various CML cell lines expressing b2a2, b3a2, T315I mutated BCR-ABL and CD34 + stem/progenitor cells from non-responder patients. Results: A total of 7 siRNA sequences targeting various fusion regions of BCR-ABL were designed and tested to effectively target primitive CML cells. The highest performing BCR-ABL siRNA encapsulated showed significant knockdown in various CML models expressing b2a2, b3a2 and T315I mutated BCR-ABL (65-99% knockdown). After 48 hours of 0.5 μg/mL BCR-ABL siRNA LNP treatment, BCR-ABL protein levels and its direct downstream target p-CrkL are undetectable by immunoblotting. The robust downregulation of BCR-ABL results in a dose-dependent decrease in cell viability (down to 28% and 9.4% viable cells at a dose of 1 μg/mL) and increase in apoptotic cells (70% and 78% Annexin-V positive) in imatinib resistant K562 and T315I mutated cells, respectively after 72 hours. Similarly, encapsulated miR-185 mimic downregulates its main downstream target PAK6 transcript levels down to 50% in drug sensitive and drug-resistant cell lines and in TKI nonresponder CD34 + stem/progenitor cells. More importantly, our RNA LNP formulation demonstrated significant uptake (> 80% Cy5 positive) of fluorescent-labelled siRNA in TKI nonresponder CD34 + stem/progenitor cells and decreased BCR-ABL mRNA transcript levels by 60%. The combined BCR-ABL inhibition and miR-185 mimic treatment were determined to be highly synergistic when analysed using the Chou-Talalay (combination index less than 1), ZIP, Bliss and HSA methods (synergy scores larger than 10) in both drug-sensitive and drug-resistant CML cells. Furthermore, treatment of BCR-ABL siRNA and miR-185 using LNPs decreased the colony forming units of nonresponder patient cells by 50%. Interestingly, this effect was further enhanced when combined with TKIs, decreasing colony forming units to 20% compared to control after 14 days. Conclusion: We have uncovered a LNP formulation that is able to effectively deliver therapeutic BCR-ABL siRNA and miR-185 mimic into primitive CML cells. This combination treatment significantly reduces the levels of oncogenic BCR-ABL and PAK6, decreasing the viability clonogenic potential and increases apoptosis in CML cell line models and primary patient cells. Importantly, this combination strategy overcomes TKI resistance in CD34 + stem/progenitor cells suggesting a feasible and effective model for patients suffering from clinical resistance and relapse.

Cell Contact with Endothelial Cells Favors the In Vitro Maintenance of Human Chronic Myeloid Leukemia Stem and Progenitor Cells

Chronic Myeloid Leukemia (CML) originates in a leukemic stem cell that resides in the bone marrow microenvironment, where they coexist with cellular and non-cellular elements. The vascular microenvironment has been identified as an important element in CML development since an increase in the vascularization has been suggested to be related with poor prognosis; also, using murine models, it has been reported that bone marrow endothelium can regulate the quiescence and proliferation of leukemic stem and progenitor cells. This observation, however, has not been evaluated in primary human cells. In this report, we used a co-culture of primitive (progenitor and stem) CML cells with endothelial colony forming cells (ECFC) as an in vitro model to evaluate the effects of the vascular microenvironment in the leukemic hematopoiesis. Our results show that this interaction allows the in vitro maintenance of primitive CML cells through an inflammatory microenvironment able to regulate the proliferation of progenitor cells and the permanence in a quiescent state of leukemic stem cells.

Assessment of Cell Cycle in Primitive Chronic Myeloid Leukemia Cells by Flow Cytometry After Coculture with Endothelial Cells.

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

Dual oligopeptides modification mediates arsenic trioxide containing nanoparticles to eliminate primitive chronic myeloid leukemia cells inside bone marrow niches.

Chronic myeloid leukemia (CML) is one type of hematopoietic stem cell diseases. Although BCR-ABL1 tyrosine kinase inhibitors are remarkably effective in inducing remission in chronic phase patients, they are not curative in a majority of patients due to their failure to eradicate residual CML stem/progenitor cells, which reside in bone marrow niches. Here, we presented novel dual oligopeptides-conjugated nanoparticles and demonstrated their effective delivery of arsenic trioxide in bone marrow niches for the elimination of primitive CML cells. We encapsulated As-Ni transitional metal compounds into polymeric nanoparticles based on the reverse micelle rationale. The loading density and stability of arsenic trioxide in nanoparticles were improved. In vitro experiments demonstrated that dual oligopeptides conjugated nanoparticles could deliver arsenic trioxide into bone marrow niches including endosteal niches and vascular niches. The colony-forming activity of CML cells was remarkably restrained in the presence of metaphyseal bone fragments pre-incubated with bone marrow niche targeted arsenic nanoparticles. The in vitro vascular niche model suggested that CML cell proliferation was also successfully inhibited through a tight contact with HUVECs, which were pre-treated using niche-targeted arsenic nanoparticles. This bone marrow niche targeted delivery strategy has a potential usage for the treatment of CML and other malignant hematologic disorders originated from the bone marrow.

A high-content cytokine screen identifies myostatin propeptide as a positive regulator of primitive chronic myeloid leukemia cells.

Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34+ CD38low chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of MSTN by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.

Similar expression profiles in CD34+ cells from chronic phase chronic myeloid leukemia patients with and without deep molecular responses to nilotinib.

The life expectancy of patients with chronic phase chronic myeloid leukemia on tyrosine kinase inhibitor therapy now approaches that of the general population. Approximately 60% of patients treated with second generation tyrosine kinase inhibitors achieve a deep molecular response, the prerequisite for a trial of treatment-free remission. Those patients unlikely to achieve deep molecular response may benefit from more intensive therapy up front. To identify biomarkers predicting deep molecular response we performed transcriptional profiling on CD34+ progenitor cells from newly diagnosed chronic phase chronic myeloid leukemia patients treated with nilotinib on a prospective clinical trial. Using unsupervised and targeted analytical strategies, we show that gene expression profiles are similar in patients with and without subsequent deep molecular response. This result is in contrast to the distinct expression signature of CD34+ chronic phase chronic myeloid leukemia patients failing to achieve a cytogenetic response on imatinib and suggests that deep molecular response to second-generation tyrosine kinase inhibitors is governed by the biology of more primitive chronic myeloid leukemia cells or extrinsic factors.

CD36 defines primitive chronic myeloid leukemia cells less responsive to imatinib but vulnerable to antibody-based therapeutic targeting.

Tyrosine kinase inhibitors (TKIs) are highly effective for the treatment of chronic myeloid leukemia (CML), but very few patients are cured. The major drawbacks regarding TKIs are their low efficacy in eradicating the leukemic stem cells responsible for disease maintenance and relapse upon drug cessation. Herein, we performed ribonucleic acid sequencing of flow-sorted primitive (CD34+CD38low) and progenitor (CD34+ CD38+) chronic phase CML cells, and identified transcriptional upregulation of 32 cell surface molecules relative to corresponding normal bone marrow cells. Focusing on novel markers with increased expression on primitive CML cells, we confirmed upregulation of the scavenger receptor CD36 and the leptin receptor by flow cytometry. We also delineate a subpopulation of primitive CML cells expressing CD36 that is less sensitive to imatinib treatment. Using CD36 targeting antibodies, we show that the CD36 positive cells can be targeted and killed by antibody-dependent cellular cytotoxicity. In summary, CD36 defines a subpopulation of primitive CML cells with decreased imatinib sensitivity that can be effectively targeted and killed using an anti-CD36 antibody.

Inhibition of Atg7 Enhances Mesenchymal Stem Cells Regulates Energy Metabolism Coupling to Overcome Drug Resistance in Chronic Myeloid Leukemia

Abstract Abstract Background and objective: A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells resistance and disease progression. Increasing researches have demonstrated that abnormal autophagy of Mesenchymal Stem Cells(MSC) becomes activated when cancer cells are subjected to chemotherapy, which is involved in the development of drug resistance. Therefore, study on the autophagy mechanism of leukemia and MSC serves as a new strategy in cancer treatment. Methods: We analyzed the biological and metabolic effect of CML primary cells and cell lines co-cultured with MSC investigate whether autophagy regulate resistance in CML cells and whether Knockdown of Atg7 by CRISPR/Cas9 could affect the viabilities of drug-sensitive and drug-resistant CML cells. Results: We detected more autophagosomes ,monocarboxylate transporter-1(MCT1) and lactic acid in CML-BC and patients with TKI-resistance significantly, and the mRNA of Atg7 showed a positive correlation with autophagosomes.In 1% oxygen concentration, Knockdown of Atg7 in KBM5-STI cells using CRISPR/Cas9 markedly decreased the level of MCT1 ,and increased apoptosis following treatment with Imatinib. Interestingly, co-cultured of KBM5-STI cells with MSC increased autophagy and chemoresistance exposed to Imatinib agents, and this was reversed following Atg7 knockdown decreased the level of MCT1 and lactic acid in MSCs. This effect was further enhanced by concomitant knockdown of Atg7 in both KBM5-STI and MSCs. Conclusions: These findings strongly suggest that microenvironment autophagy regulates energy metabolism coupling and plays an important role in CML chemoresistance.Thus, the inhibition of Atg7 appears to be a valid strategy to enhance TKI-sensitivity, and it could indeed improve outcomes in CML therapy Disclosures No relevant conflicts of interest to declare.

Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells.

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second/third generation c-Abl specific tyrosine kinase inhibitors (TKIs) has significantly extended patient survival1. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs)2–4. Therefore, targeting minimal residual disease, to prevent acquired resistance and/or disease relapse requires identification of novel LSC-selective target(s) that can be exploited therapeutically5,6. Given that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner7, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34+ and CD34+CD38-) and differentiated (CD34-) patient derived CML cells, and compared their signature with that of normal counterparts. Combining stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination-treatment of imatinib with tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs, both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong indication for investigating the employment of TKIs in combination with tigecycline to treat CML patients with minimal residual disease.

A novel AHI-1\u2013BCR-ABL\u2013DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

Tyrosine kinase inhibitor (TKI) therapies induce clinical remission with remarkable effects on chronic myeloid leukemia (CML). However, very few TKIs completely eradicate the leukemic clone and persistence of leukemic stem cells (LSCs) remains challenging, warranting new, distinct targets for improved treatments. We demonstrated that the scaffold protein AHI-1 is highly deregulated in LSCs and interacts with multiple proteins, including Dynamin-2 (DNM2), to mediate TKI-resistance of LSCs. We have now demonstrated that the SH3 domain of AHI-1 and the proline rich domain of DNM2 are mainly responsible for this interaction. DNM2 expression was significantly increased in CML stem/progenitor cells; knockdown of DNM2 greatly impaired their survival and sensitized them to TKI treatments. Importantly, a new AHI-1–BCR-ABL–DNM2 protein complex was uncovered, which regulates leukemic properties of these cells through a unique mechanism of cellular endocytosis and ROS-mediated autophagy. Thus, targeting this complex may facilitate eradication of LSCs for curative therapies.

The Focal Adhesion Complex Ilk-Pinch-Parvin Is Key to Niche Interactions and Survival of Quiescent Stem Cells in Chronic Myeloid Leukemia In Vitro and In Vivo

Growing evidence indicates that interactions of cancer cells with their microenvironment in vivo can influence disease progression and therapy resistance, including chronic myeloid leukemia (CML). Focal adhesions that modulate cell attachments, migration, proliferation and intracellular signaling pathways are considered critical mediators of some of these interactions. However, the potential role of focal adhesion components in mediating survival and therapeutic responses of leukemic stem cells is largely unknown.Transcriptional profiling of CD34+ cells from 6 CML patients and 3 healthy donors revealed that the expression of Integrin-linked kinase (ILK), PINCH1 and β-Parvin, major constituents of focal adhesions, is significantly increased in CD34+ CML cells, in particular in cells from drug-nonresponders (p<0.05). Quantitative real-time PCR confirmed these observations in CD34+ cells obtained from additional 30 CML patients and 14 normal healthy adults (p<0.05). Furthermore, we found that the primitive leukemic stem-cell enriched Lin-CD34+CD38- portion from CML patients expressed the highest levels of ILK, PINCH1, and β-Parvin transcripts compared to the more prevalent Lin-CD34+CD38+ progenitor population or mature CD34-cells in the same samples (n=6, p<0.05). In addition, ILK protein expression was increased in primitive CML cells compared to normal donors, in particular when CML cells were co-cultured with BM niche cells.Stable knockdown (KD) of 3 different targeting sequences of ILK in CD34+ CML cells resulted in decreased cell viability (30-80%, p<0.05) and proliferation (2-12-fold) associated with a significantly enhanced frequency of apoptotic cells compared to control-transduced cells (60-80% vs. 30%, p<0.05). Interestingly, these effects of ILK KD were not rescued by co-cultures with BM niche cells in vitro. Cell cycle analysis indicated a reduction in the proportion of surviving cells in S-phase upon ILK suppression. In addition, Western blotting showed that effective suppression of ILK led also to a decrease in β-Parvin and PINCH1 protein expression but not their transcript levels, suggesting that the ILK-PINCH-PARVIN complex is not stable under these conditions and may not be able to mediate critical interactions between primitive CML cells and BM niche components. In agreement, short- and long-term assays of stem/progenitor activity in the presence of BM niche cells demonstrated a significant reduction of colonies upon ILK suppression that was almost entirely abolished with simultaneous ABL1 tyrosine kinase inhibitor (TKI) treatment (p<0.05). Moreover, in vivo studies with 2 different mouse strains (NRG and the humanized NRG-3GS model) emphasized that primitive ILK KD CML cells showed greatly reduced in vivo regenerative activity as compared to control-transduced cells (<2% vs. 13% human cells in the BM of NRG mice, and 3% vs. 18% in NRG-3GS mice 25 weeks post-transplantation).To investigate whether ILK can be targeted pharmacologically, we utilized QLT0267, a validated and selective ILK kinase inhibitor. Similarly to ILK suppression, inhibition of the ILK kinase resulted in a modest decrease of cell viability, reduced short-and long-term stem/progenitor activity, and increased apoptosis of bulk CD34+ as well as more primitive Lin-CD34+CD38- CML cells from drug-nonresponder patients with strong synergistic effects upon simultaneous ABL1 kinase inhibition in vitro. In addition, oral gavage of QLT0267 combined with dasatinib significantly enhanced survival of leukemic mice and eradicated infiltrated leukemic cells in multiple hematopoietic tissues in an aggressive NSG mouse model of BCR-ABL+human leukemia. Most interestingly, dual inhibition of ILK and BCR-ABL1 decreased the proportion of quiescent leukemic stem cells compared to single agent treatments. RNA sequencing of these cells indicated a deregulation of MYC and novel signaling targets, with differences between dividing and non-dividing cell subpopulations.In summary, genetic and pharmacological inhibition of ILK significantly impaired survival, proliferation and quiescence of drug-nonresponder CML stem cells and sensitized them to TKIs both in vitro and in vivo. These findings suggest that ILK plays a critical role in regulating CML stem cell activity and that targeting ILK and BCR-ABL1 simultaneously may offer an improved novel therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

IL1RAP Antibodies Block IL1-Induced Expansion of Primitive CML Cells and Display Therapeutic Effects in Xenograft Models

Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML.We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells.Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells.To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2.We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. DisclosuresRichter:BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

ABSTRACTA major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.

Discovery of survival factor for primitive chronic myeloid leukemia cells using induced pluripotent stem cells

A definitive cure for chronic myeloid leukemia (CML) requires identifying novel therapeutic targets to eradicate leukemia stem cells (LSCs). However, the rarity of LSCs within the primitive hematopoietic cell compartment remains a major limiting factor for their study in humans. Here we show that primitive hematopoietic cells with typical LSC features, including adhesion defect, increased long-term survival and proliferation, and innate resistance to tyrosine kinase inhibitor (TKI) imatinib, can be generated de novo from reprogrammed primary CML cells. Using CML iPSC-derived primitive leukemia cells, we discovered olfactomedin 4 (OLFM4) as a novel factor that contributes to survival and growth of somatic lin−CD34+ cells from bone marrow of patients with CML in chronic phase, but not primitive hematopoietic cells from normal bone marrow. Overall, this study shows the feasibility and advantages of using reprogramming technology to develop strategies for targeting primitive leukemia cells.

Role of Stem Cell Elements in Chronic Myeloid Leukemia

Context: Chronic myeloid leukemia (CML) is believed to occur following the clonal expansion of haematopoietic stem cells and is maintained by expanding clones which have acquired a BCR-ABL fusion gene. The properties of untreated CML stem/progenitor cells correlate with a subsequent response to chemotherapy. Evidence Acquisition: The fifty two significant articles discussing the stem cell in chronic myeloid leukemia from 1996 to 2012 were selected according to the authors’ experience. Results: Studies have shown that primitive CML cells are less responsive to Tyrosine Kinase Inhibitors (TKIs) and are a reservoir for the relapse of multi drug resistant (MDR). Conclusions: Following that, minimal residual disease (MRD) measurement aims to detect very small numbers of leukemic cells, below the detection limit of morphology and cytogenetics with molecular techniques for patients in clinical remission.

Integrin-Linked Kinase As a Key Mediator of Stromal Cell-Enhanced Resistance of Primitive CML Cells to Tyrosine Kinase Inhibitors

The human bone marrow (BM) compartment consists of a heterogeneous, multi-functional network of cells and extracellular matrix that interact with hematopoietic stem cells. Growing evidence suggests that this microenvironment also likely adds to the resistance of chronic myeloid leukemia (CML) stem cells to tyrosine kinase inhibitor (TKI) therapy in vivo. Integrin-linked kinase (ILK) is a serine/threonine kinase that is an important constituent of focal adhesions, a regulator of spindle formation during mitosis, and a key mediator of multiple signaling pathways. However, its potential role in the regulation of primitive CML cells and their response to TKIs is unknown.Our RNA-seq analysis revealed that ILK expression and its downstream targets, such as AKT, are highly upregulated in pre-treatment CD34+ CML stem/progenitor cells (n=6) compared to normal BM controls (n=3, p<0.05). We confirmed this finding by qRT-PCR analysis of CD34+ cells obtained from 28 CML patients (including some who subsequently became clinically resistant to imatinib mesylate (IM) treatment) compared to CD34+ BM cells from 9 normal adults (p<0.05). Elevated expression of ILK protein in CD34+ CML cells was also demonstrated by Western blot analysis (threefold, n=4, p<0.05). Interestingly, we also found ILK transcript levels to be much higher in the more primitive and rarer CD38- CML stem cell-enriched subset of the lin-CD34+ population as compared to the more mature and prevalent CD38+ subset or the terminally differentiated lin+CD34- cells (p<0.01). Both ILK transcripts and intracellular protein levels were elevated in CD34+ CML cells, but not in CD34- differentiated cells, in response to IM treatment in vitro, especially in the presence of expanded BM stromal cells, suggesting differential regulation of ILK expression in primitive CML cells and their more mature counterparts.We next asked whether ILK influences primitive CML cell responses to TKIs by using a clinically validated and selective ILK inhibitor (QLT0267) to suppress ILK activity in colony assays of CD34+ CML cells. QLT0267 plus TKIs significantly reduced the yield of colonies obtained compared to any single agent or combination of TKIs and this enhanced cell killing was most pronounced on cells from IM non-responders (n=6; p<0.01). Analysis of the lineages affected showed that the combination of IM or dasatinib (DA) with QLT0267 had a more pronounced effect on myeloid colony formation (CFU-GM), at concentrations where either or both TKIs had little effect (i.e. 85-90% inhibition vs. 30-45%). Moreover, the simultaneous suppression of ILK and BCR-ABL activities also effectively inhibited the growth of more primitive CML cells (from IM non-responder patients) when these were co-cultured with stromal cells in 6-week long-term cultures (LTC), in contrast to the lack of these effects in the presence of single TKIs or TKI combinations.Mechanistically, we found that the combination of QLT0267 with a TKI enhanced the induction of apoptosis of CD34+ CML cells in suspension cultures within the first three days (from 10-15% to 30-45% apoptotic cells relative to untreated controls) and CFSE (carboxy-fluorescein diacetate succinimidyl diester) tracking of cell division showed that QLT0267 specifically targets quiescent CML stem cells from IM-resistant patients (n=4; p<0.05). In addition, treatment with the ILK inhibitor alone or in combination with a TKI enhanced apoptosis of primitive CML cells in vitro by abolishing the protective effect of BM stromal cells observed under TKI treatment alone (25-30% apoptotic cells vs. 3-8%) and combination treatments confirmed strong synergy between DA and QLT0267 (CI value <0.3). Importantly, QLT0267 (up to 10 μM) was not toxic to normal CD34+BM cells in either short- or long-term culture systems (with and without stromal cells).Together, these findings indicate that ILK is a critical player in the regulation of TKI response/resistance of primitive CML cells. The enhanced and selective effect obtained by dual inhibition of both targets, particularly in the presence of protective stromal cells to mimic their response within the BM microenvironment, may offer an important new therapeutic possibility. DisclosuresNo relevant conflicts of interest to declare.

Over-Expression of Heme Oxygenase-1 in Peripheral Blood Predicts the Progression and Relapse Risk of Chronic Myeloid leukemia

Background There are limited eligible clinical markers at present to monitor the progress of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1), as one of the most important oxidation-regulating enzymes in vivo, suggests the onset and progression of cancer when highly expressed. Furthermore, HO-1 level is related with the occurrence and development of hematological diseases. But the relationship between HO-1 expression and progression/relapse of CML has seldom been studied hitherto. This study aimed to investigate the relationship between them to find out a new molecular marker for prediction.Methods A total of 60 peripheral blood and bone marrow (BM) samples from 25 CML patients in different phases were collected respectively to detect the expressions of HO-1 and bcr/abl using real-time PCR. Routine blood test was performed to detect the changes of leukocyte and platelet counts. The proportion of primitive cells in BM was detected by flow cytometry. The relationship between high HO-1 expression and CML progression and relapse was explored by the analysis of variance by Wilcoxon test and linear regression analysis. The diagnostic accuracy and cutoff values were determined by receiver operating characteristic curve.Results Relative expression of HO-1 mRNA in CML patients peripheral blood was significantly higher than that of donors (P <0.0001), which were 0.57±3.78 and (1.417±1.125)×10–6, respectively. HO-1 expression level in CML patients was 0.061 5±0.062 4, which decreased to 0.009 4±0.006 7 upon CMoR, and remained remarkably higher 0.016 3±0.017 5 than that of normal donors (1.417±1.125)×10–6, P <0.001. When relapse occurred, HO-1 expression significantly increased from 0.020 6±0.021 0 to 3.852±10.285 in CMoR stage and undergoing relapse. According to progression of CML, HO-1 expression level in CML patients increased from CP (0.009 5±0.017 6) to AP (0.028 0±0.055 7) and then to BP (0.276 7± 0.447 0) . And there was a linear correlation between HO-1 expression and proportion of primitive CML cells. The diagnostic accuracies and cutoff values of HO-1 expression for CML-CP, CML-AP, and CML-BP were 1.0, 0.748, and 0.965, respectively, as well as 0.000 070, 0.001 917, and 0.020 696, respectively.Conclusion HO-1 may be a potential molecular indicator for the progression and relapse of CML. DisclosuresNo relevant conflicts of interest to declare.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Whole Genome Sequencing Of Chronic Myeloid Leukemia (CML)-Derived Induced Pluripotent Stem Cells (iPSC) Reveals Faithful Genocopying Of Highly Mutated Primary Leukemic Cells

Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient's CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 105 cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data. Disclosures:Turhan:BMS, Novartis: Honoraria, Research Funding.

Effectively Targeting Treatment-Naïve CML Stem/Progenitor Cells From Imatinib-Nonresponders With Combination Treatments Of JAK2 and ABL Inhibitors In Vitro and In Vivo

The hallmark of chronic myeloid leukemia (CML) is the presence of a BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. Introduction of Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies has had a major impact on the treatment of chronic phase CML, but early relapses and persistence of leukemic stem cells remain problematic. We have recently identified an AHI-1–BCR-ABL–JAK2 protein complex that contributes to the transforming activity of BCR-ABL and to IM-resistance of CML stem/progenitor cells. We therefore hypothesized that combined suppression of BCR-ABL and JAK2 activities might more effectively eliminate CML stem/progenitor cells in vitro and in vivo. Several JAK2 inhibitors are currently in various stages of clinical trials, but their off-target effects on normal primitive hematopoietic cells remain a concern. We have now examined the biological effects of an orally bioavailable, selective JAK2 inhibitor (BMS-911543) in combination with TKIs, including IM, dasatinib (DA) and nilotinib, both on CML cells lines and CD34+ treatment-naïve IM-nonresponder cells, which were obtained at diagnosis from CML patients who were classified subsequently, after initiation of IM therapy, as IM-nonresponders. In cell line studies, Western blot analysis showed that combination treatment was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells than single agents. In colony-forming cell (CFC) assays, combination treatment resulted in a greater reduction in colonies produced from these cells compared to single agents (2-3 fold, p<0.05). Similarly, intracellular staining analyses showed that combined exposure of CD34+ CML cells (n=4) to BMS-911543 and a TKI produced a deeper, more prolonged suppression of pSTAT5 and pCRKL activity than single agents (40-46% suppression for the combinations vs. 15-20% suppression for the single agents at 72 hrs, p<0.05). Combination treatments also resulted in greater inhibition of colony growth of CD34+ CML cells compared to single agents (n=7, 74-86% vs. 40-50%, p<0.05). Interestingly, the combination of BMS-911543 and a TKI almost completely inhibited BFU-E colony formation as compared to treatment with TKI alone (92-100% vs.63-66%, p<0.01). CFU-GM colonies were also more significantly reduced as a result of combination treatment, compared to single agents (49-71% vs. 30-39%, p<0.01). Long-term culture-initiating cell assays showed that more primitive cells were also more significantly eliminated by combination treatments (n=3, 2-3 fold, p<0.05). Importantly, our CFC data indicate that BMS-911543 is less toxic to normal bone marrow (BM) CD34+ cells (n=7) than the same cells from CML samples (n=7, 2-3 fold, p<0.05). To test the ability of combination treatments to eliminate primitive CML cells with in vivo leukemia propagating activity, we injected primitive CML cells intravenously into NSG mice and treated mice with inhibitors by oral gavage for two weeks. Mice undergoing combination treatment showed significantly reduced weight loss and engraftment levels in peripheral blood, BM , spleen, and liver compared to mice treated with single agents (0.14% vs. 6.14%, 1.17% vs. 26.3%, 2.46% vs. 51.5%, and 77.8% vs. 92.9%, respectively, p<0.05). H&E histology staining revealed that mice treated with DA and BMS-911543 had less infiltration of leukemic cells into their spleens and livers than mice treated with DA alone. Quantitative RT-PCR analysis further demonstrated a statistically significant reduction in BCR-ABL transcript levels in spleen, liver, and BM of mice treated with a combination of DA and BMS-911543 compared to mice treated with single agents (15-20 fold, p<0.05). Most importantly, combination treatments significantly enhanced survival of leukemic mice compared to mice treated with single agents (median survival of IM + BMS-911543 vs. IM: 70 days vs. 60.5 days, p<0.05; DA + BMS-911543 vs. DA: 96.5 days vs. 81 days, p<0.001). This study suggests that simultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients, especially those destined to develop TKI resistance. Disclosures:No relevant conflicts of interest to declare.