BCR-ABL Independent Mechanisms Research Articles

Molecular Pathogenesis and Treatment Strategies of Chronic Myeloid Leukemia (CML)

Chronic Myeloid Leukemia (CML) is a myeloproliferative disease diagnosed in bone marrow, arising from a chromosomal translocation between chromosomes 9 and 22, resulting in the formation of fusion oncogene BCR–ABL. The product of this fusion oncogene is a new oncoprotein bcr–abl which possesses abnormal tyrosine kinase activity. In response to this, abnormal signaling pathway activation occurs, leading to cell transformation. BCR–ABL oncogene could be targeted by tyrosine kinase inhibitors (TKIs) to delay or inhibit the disease progression. Imatinib is the first drug designed against CML but resistance to this has led to the development of the second- and third generations of inhibitors that are active against many types of BCR–ABL gene mutations. However, somehow, due to disease progression, TKIs do not remain as effective. There are three well-characterized phases of CML: The chronic phase (CP), the accelerated phase, and the terminal stage which is the blast crisis (BC) stage. In the CP of CML, mature granulocytes and myeloid precursors become aggregated majorly in the bone marrow and peripheral blood. The accelerated phase is marked by increased disease severity and an increase in progenitor/precursor cell number. In the BC stage, undifferentiated blast cells grow in number. Many patients with CML are diagnosed during the CP of the disease, so the survival rate of CML is high. However, 20% of CML patients proceed to advanced stages that result in drug resistance, intolerance, and mortality. So, for proper CML treatment, drugs are needed to target multiple BCR– ABL mutations, delay or stop disease progression, and overcome resistance caused by BCR–ABL independent mechanisms, especially during advanced phases of CML. Moreover, drugs could be developed to eradicate the stem cells of CML. These targets could be achieved by understanding mechanisms of disease progression, disease relapse, and drug resistance by utilizing high throughput molecular genetics, cell biology and immunology techniques.

BCR-ABL Independent Resistance Mechanisms to Chronic Myeloid Leukemia

BCR-ABL Independent Resistance Mechanisms to Chronic Myeloid Leukemia

Intracellular angiopoietin-1 promotes TKI-resistance via activation of JAK/STAT5 pathway in chronic myeloid leukemia.

Drug resistance from BCR-ABL tyrosine kinase inhibitors (TKIs) and other chemotherapeutics results in treatment failure and disease progression in chronic myeloid leukemia (CML). However, the mechanism is still uncertain. In this study, we investigated the role of angiopoietin-1 (ANG-1) as a potential prognostic factor for drug resistance in CML. Both intracellular and secretory ANG-1 (iANG-1 and sANG-1) were overexpressed in multidrug-resistant CML samples. The IC50 value was higher in primary CD34+ CD38- cells with more ANG-1. Silencing ANG-1significantly sensitized three TKI-resistant CML cell lines to imatinib (IM) while recombinant human ANG-1 failed to retain cell survival in vitro. This indicated the important role of iANG-1 as opposed to sANG-1 in CML drug resistance. Moreover, a similar effect was observed in xenograft mice models bearing ANG-1-silenced CML cells. Subsequently, pathway analysis and protein validation experiments showed activation of the JAK/STAT pathway and augmentation of STAT5a phosphorylation in ANG-1 restored CML cells. Upstream Src phosphorylation, which plays a crucial role in CML drug resistance, was also upregulated as a key event in iANG-1-related JAK/STAT pathway activation. In conclusion, our study elucidated a new BCR-ABL independent molecular mechanism induced by intracytoplasmic ANG-1 overexpression as a potential strategy for overcoming CML resistance.

CD44: a potential therapeutic target in chronic myeloid leukemia.

Development of tyrosine kinase inhibitors (TKIs) achieved great success in the treatment of chronic phase chronic myeloid leukemia (CML). However, patients with CML still relapse without taking TKIs and cases in the accelerated phase or aggressive blast crisis rarely achieved deep response to TKIs. Drug resistance and persistence of leukemia stem cell (LSC) remain great challenges. BCR-ABL kinase dependent or independent mechanism of action are still far from being understood. To achieve a stable deep molecular response and treatment-free remission, finding new targets, eliminating LSC, reducing recurrence and improving prognosis are problems urgently to be solved. It is revealed that tumor microenvironment is crucial for survival, invasion and metastasis of tumor cells. As an adhesion molecule, CD44, a single-chain transmembrane glycoprotein, is not only being identified as a marker for cancer stem cells, but also plays a crucial role in microenvironmental communication and transmitting intracellular signaling for cell proliferation, differentiation, migration, and contributes to tumorigenesis. In this review, we focus on current data relevant to CD44, and outline CD44 structure, the regulation of CD44, functional properties of CD44 in survival, resistance, CML stem cells as well as the potential CD44-targeting therapy for CML management.

Abstract 5267: Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells

Abstract Ponatinib is a clinically approved tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. In this study stimulated Raman scattering (SRS) microscopy was used to allow label-free imaging of intracellular ponatinib with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. SRS microscopy represents a powerful imaging tool for visualizing label-free drug molecules within cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing the intracellular concentrations of drug molecules to be accurately determined. Intracellular visualization of ponatinib was achieved at biologically relevant, nanomolar concentrations for the first time using SRS. It was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not re-sensitise cells to ponatinib, confirming BCR-ABL independent resistance mechanism in this CML cell model. In summary, we have visualized intracellular ponatinib localization for the first time using SRS, demonstrating that acquired drug resistance can influence drug uptake and localisation in CML, which in turn has an effect on target engagement. Citation Format: Kristel Sepp, Martin Lee, Marie T. Bluntzer, G. Vignir Helgason, Alison N. Hulme, Valerie G. Brunton. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5267.

BCR-ABL Independent Mechanisms of Resistance in Chronic Myeloid Leukemia

Not all chronic myeloid leukemia (CML) patients are cured with tyrosine kinase inhibitors (TKIs), and a proportion of them develop resistance. Recently, continuous BCR-ABL gene expression has been found in resistant cells with undetectable BCR-ABL protein expression, indicating that resistance may occur through kinase independent mechanisms, mainly due to the persistence of leukemia stem cells (LSCs). LSCs reside in the bone marrow niche in a quiescent state, and are characterized by a high heterogeneity in genetic, epigenetic, and transcriptional mechanisms. New approaches based on single cell genomics have offered the opportunity to identify distinct subpopulations of LSCs at diagnosis and during treatment. In the one hand, TKIs are not able to efficiently kill CML-LSCs, but they may be responsible for the modification of some LSCs characteristics, thus contributing to heterogeneity within the tumor. In the other hand, the bone marrow niche is responsible for the interactions between surrounding stromal cells and LSCs, resulting in the generation of specific signals which could favor LSCs cell cycle arrest and allow them to persist during treatment with TKIs. Additionally, LSCs may themselves alter the niche by expressing various costimulatory molecules and secreting suppressive cytokines, able to target metabolic pathways, create an anti-apoptotic environment, and alter immune system functions. Accordingly, the production of an immunosuppressant milieu may facilitate tumor escape from immune surveillance and induce chemo-resistance. In this review we will focus on BCR-ABL-independent mechanisms, analyzing especially those with a potential clinical impact in the management of CML patients.

Abstract 2106: Role of organic cation transporter-1 (OCT-1) in drug resistance condition of ph+ chronic myeloid leukemia (CML)

Abstract A solute carrier (SLC) superfamily member, a major influx protein Organic Cation Transporter-1 (OCT-1), reported to be involved in influx of the Imatinib and other drugs in target cells. Despite the excellent responses to Imatinib and other tyrosine kinase inhibitors (TKIs) in CML, around a quarter of patients demonstrate primary resistance or suboptimal response within one year of therapy. Hence, present study mainly emphases the possible translocation mechanism of Imatinib, major TKIs and other chemotherapeutic agents that is mediated by OCT-1 transporter and OCT-1 gene expression level in progressive phases of CML. Homology based comparative modeling of OCT-1 protein, and its in-depth systemic interaction binding pattern analysis with Imatinib and other TKIs were conducted using computational modeling and molecular dynamics studies, respectively. Quantitative expression analysis of OCT-1 has also been evaluated in patients receiving Imatinib alone, hydroxyurea alone or both drug in different CML phases such as chronic, accelerated and blastic crisis phases for both in bone marrow and PBMNC samples (n=120). The interaction profile of Imatinib with OCT-1 revealed major conformational between ligand and protein interaction sites. However, generated protein-ligand complexes proceeded for molecular dynamics studies (100ns) that have confirmed the selectivity of Imatinib and other drugs towards the OCT-1 and possible translocation path of ligands in the OCT-1 cavities. In quantitative gene expression analysis, downregulation of OCT-1 expression as evident by decreased fold change values in patients with blastic crisis compared to chronic phase responsible for lower uptake of Imatinib into the cells. Hence, cells can not achieve optimum Imatinib inhibitory concentration which may be responsible for disease progression and ultimately leads to drug resistance. Thus, proposed combined computational and experimental approaches suggests OCT-1 mediated activation of bcr-abl independent mechanism, which may be potential confounder of developing Imatinib resistance in CML patients. Citation Format: Saumya Patel, Krupa Shah, Rakesh Rawal. Role of organic cation transporter-1 (OCT-1) in drug resistance condition of ph+ chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2106.

Aberrant DNA methylation at HOXA4 and HOXA5 genes are associated with resistance to imatinib mesylate among chronic myeloid leukemia patients.

Imatinib mesylate is a molecularly targeted tyrosine kinase inhibitor drug. It is effectively used in the treatment of chronic myeloid leukemia (CML) patients. However, development of resistance to imatinib mesylate as a result of BCR-ABL dependent and BCR-ABL independent mechanisms has emerged as a daunting problem in the management of CML patients. Between these mechanisms, BCR-ABL independent mechanisms are still not robustly understood. To investigate the correlation of HOXA4 and HOXA5 promoter DNA hypermethylation with imatinib resistance among CML patients. Samples from 175 Philadelphia positive CML patients (83 good response and 92 BCR-ABL non-mutated imatinib resistant patients) were subjected to Methylation Specific High Resolution Melt Analysis for methylation levels quantification of the HOXA4 and HOXA5 promoter regions. Receiver operating characteristic curve analysis was done to elucidate the optimal methylation cut-off point followed by multiple logistic regression analysis. Log-Rank analysis was done to measure the overall survival difference between CML groups. The optimal methylation cut-off point was found to be at 62.5% for both HOXA4 and HOXA5. Chronic myeloid leukemia patients with ≥63% HOXA4 and HOXA5 methylation level were shown to have 3.78 and 3.95 times the odds, respectively, to acquire resistance to imatinib. However, overall survival of CML patients that have ≤62% and≥63% methylation levels of HOXA4 and HOXA5 genes were found to be not significant (P-value=0.126 for HOXA4; P-value=0.217 for HOXA5). Hypermethylation of the HOXA4 and HOXA5 promoter is correlated with imatinib resistance and with further investigation, it could be a potential epigenetic biomarker in supplement to the BCR-ABL gene mutation in predicting imatinib treatment response among CML patients but could not be considered as a prognostic marker.

Characterization of imatinib-resistant K562 cell line displaying resistance mechanisms

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.

High Peroxisome Proliferator-Activated Receptor-Gamma (PPARγ) Transcriptional Activity Reduces Active Influx of Imatinib and Kinase Inhibition in CML Cells

▪The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL1+ cells. A low functional OCT-1 activity (OA) in peripheral blood mononuclear cells (MNCs) at diagnosis is associated with poorer subsequent molecular response and inferior outcomes in chronic phase chronic myeloid leukaemia (CP-CML) patients. We have previously demonstrated that diclofenac, an antagonist of peroxisome proliferator-activated receptor-γ (PPARγ), can significantly increase OA in BCR-ABL1+ cells. In the current study, we systematically assess the correlation between PPARγ and OA using leukemic cell lines and primary MNCs from untreated de novo CP-CML patients. Given the critical role of PPARγ in cell differentiation and the varying levels of PPARγ expression in different cell types, the present study also explored the relationship between the OA and myeloid-specific markers (CD14, CD15 and CD16) in CP-CML patient MNCs.Our results demonstrate that OA in BCR-ABL1 positive and negative leukemic cell lines (KU812, HL60 and HL60-BCRABL1) were significantly decreased with PPARγ agonists (GW1929, rosiglitazone) and increased with antagonists (GW9662, T0070907) (Table 1). Similarly, OA in PPARγ-transduced K562 cells (16.03 ng/200,000 cells) was significantly reduced when compared with empty vector control (22.73 ng/200,000 cells, p=0.028). In primary MNCs isolated from untreated CP-CML patients with high OA, a consistent inhibitory effect on OA was observed following the treatment with PPARγ agonists in all 5 cases. The average OA was significantly reduced by GW1929 (from 7.88 to 5.21 ng/200,000 cells, p=0.0075) or rosiglitazone (to 4.65 ng/200,000 cells, p<0.0001). In contrast, following treatment with PPARγ antagonists, OA was increased in all but one of the low OA patient samples (n=6). The average OA in these patients was increased from 2.71 to 5.07 ng per 200,000 cells by GW9662 (p=0.0129) and to 4.71 ng per 200,000 cells by T0070907 (p=0.0155). In addition, in combination with imatinib, these effects on OA resulted in corresponding changes in Bcr-Abl kinase inhibition and cell viability. While there was no correlation between OA and the expression of the PPARG gene or total PPARγ protein, there was a negative association between OA and PPARγ transcriptional activity in MNCs samples isolated from 84 untreated CP-CML patients (r=-0.5677, p<0.0001, Figure 1). Interestingly, in primary CML samples, the percentage of CD15+ CD14- CD16high MNC cells was significantly higher in patients with low OA (41.70%, n=10) than those with high OA (21.74%, n=11, p=0.0126), which may contribute to the higher PPARγ activity observed in these cases.In summary, the findings presented in this study highlight for the first time the association between active influx of imatinib and PPARγ. The consistent OA changes in both in BCR-ABL1 positive and negative leukemic cell lines indicate that treatment with PPARγ ligands significantly alter OA via a likely Bcr-Abl independent mechanism. In CP-CML patients at diagnosis, there is a strong negative correlation between OA and PPARγ activity. By up-regulating OA, PPARγ antagonists significantly increased intracellular imatinib and Bcr-Abl kinase inhibition, that subsequently resulted in increased imatinib induced apoptosis. Therefore, reagents repressing PPARγ activity may improve responses for de novo CP-CML patients with low OA. In addition, the CD15+ CD14- CD16high subset of MNCs is significantly enriched in low OA samples providing a potential readily translatable biomarker for a patientÕs molecular response to imatinib.Table 1OCT-1 activity changes in cell lines and MNC from CP-CML patients after treatment with PPARγ ligands (percentage of vehicle control)PPARγ agonistsPPARγγ antagonistGW1929RosiglitazoneGW9662T0070907KU81267%**49%**139%**151%**HL6075%**77%*123%*123%*HL60-BCRABL174%*79%*118%*136%***, p<0.05; **, p<0.01; compared with vehicle control. [Display omitted] DisclosuresHughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Patients with Chronic Myeloid Leukemia Show Different Modulation of MYB-Dependent Oncogenic Pathway in the Course of Hematopoietic Differentiation upon Sensitivity to the TKI Treatment

Introduction: Inhibition of the BCR-ABL protein activity by tyrosine kinase inhibitors (TKIs) plays crucial role in leukemic transformation from the chronic phase (CP) of the chronic myeloid leukemia (CML) into fatal blast crisis (BC). However 20 - 30 % of CML patients develop resistance to the TKIs. Beside mutations in BCR-ABL kinase domain, BCR-ABL independent mechanisms of acquired TKIs resistance including microRNAs (miR) may be also involved. A significant reduction of miR-150 levels associates with CML progression. MiR-150 is an inhibitor of the Myeloblastoma oncogene c-MYB, which is required for BCR-ABL-dependent leukemogenesis in a mouse model of CML-BC. Recently MYB was found to positively regulating another miR, miR-155, that inhibits tumor suppressor and pro-differentiation factor PU.1 in AML/MPS mouse model. PU.1 is a major regulator of myelopoiesis whose levels require precise guidance. We recently reported increased levels of miR-155 in CML-BC.Objectives: We hypothesize that BCR-ABL activity together with low levels of miR-150 activates putative oncogenic signaling pathway MYB/miR-155/PU.1 in CML leading to a progressive blockade of cell differentiation and possibly also of resistance to TKIs.Material and Method: Bone marrow (BM) cells of CML-CP patients at the time of diagnosis (n=10), and of treated patients either sensitive (n=8) or resistant (n=11) to TKIs, were FACS sorted for CD34 and CD38 into subpopulations representing the stages of cell differentiation. CD34+ and CD34- leukocytes from peripheral blood of healthy donors (n=10) were isolated by MACS and used as control. Levels of miR-150, BCR-ABL, MYB, miR-155 and PU.1 were determined by qRT-PCR. Expression of surface differentiation markers on BM cells was determined using flow cytometry at the time of diagnosis (n=10) and upon TKIs resistance (n=5). The effect of miR-150 overexpression and BCR-ABL inhibition was studied in CML-BC cell line K562 using nucleofection of the synthetic miR-150 and siRNA BCR-ABL (and by cell cultivation with TKI imatinib). mRNA and protein expression of BCR-ABL, MYB and miR-150 levels were determined by qRT-PCR and immunoblotting.Results: In the groups of healthy donors and de novo diagnosed CML patients we observed significant positive or negative correlations of expression levels between studied molecules among the sorted cell subpopulations, which outlined the activity of the putative pathway BCR-ABL/miR-150/MYB/miR-155/PU.1 in CML-CP. Interestingly, in the group of patients resistant to TKI, we found that correlation between MYB and miR-155 was not observed suggesting this pathway is deregulated upon TKI resistance. Conversely, we found gain of negative correlation between expression of MYB and PU.1 in the group of patients responding to TKI suggesting that MYB and PU.1 oppose each other upon normal cell differentiation. We also observed significant differences of the surface marker phenotype profiles between BM cells of de novo diagnosed CML patients and patients resistant to TKIs treatment. While relatively differentiated (granulocyte) CD34- CD38- CD11b+ CD14- population presents about 7 % of BM cells for the group of patients at diagnosis, it comprises only 0.3 % of BM cells for resistant group (P= 0.0027) indicating that TKI resistance associates with significant blockade of myeloid differentiation.We next utilized K562 cells showing that levels of miR-150 significantly increased after BCR-ABL inhibition by imatinib (P=0.0017) and also upon BCR-ABL knockdown with siRNA (P=0.002). This suggests that miR-150 is regulated by BCR-ABL. Similarly, MYB mRNA and protein levels were markedly decreased upon miR-150 overexpression and this was enhanced by inhibition of BCR-ABL activity with imatinib.Conclusions: Dysregulation of BCR-ABL/miR-150/MYB/miR-155/PU.1 pathway was observed in CML stages and stage-specific cell populations in relation to TKI-sensitivity and disease progression. Our model consists of the BCR-ABL upregulating MYB both through miR-150 inhibition and by other downstream mechanisms by additive effect, thus the cooperation of miR-150 and imatinib through inhibiting MYB may represent an important barrier to CML progression. Even more our results show that regulation of miR-155 and PU.1 may play role in resistance to TKI treatment.Supported by GAUK 178215 and by project 00023736 from the Czech Ministry of Health DisclosuresKlamova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Interferon gamma modulates sensitivity of CML cells to tyrosine kinase inhibitors

Immune effector cells such as T and NK cells can efficiently eliminate tumor cells. However, when activating oncogenic signaling pathways or protective mechanisms against cell death are active, immune cells can also confer therapy resistance. Here, we analyzed the role of activated T and NK cells and released cytokines on tyrosine kinase inhibitors imatinib and nilotinib – mediated apoptosis induction and proliferation of chronic myelogenous leukemia (CML) cells. Incubation of CML cells with activated, but not with resting CD3+ T cells or with activated NK cells significantly inhibited TKI-induced apoptosis induction in CML cells as quantified by nuclear fragmentation assays. Transwell experiments revealed a critical role for T or NK cell-derived cytokines for CML cell protection. Accordingly, CML cells treated with IFNγ also showed a clearly reduced sensitivity to TKI-mediated cell death induction and inhibition of proliferation. In contrast, IFNα or other pro-inflammatory mediators and cytokines, such as TNFα and GM-CSF did not impair TKI-induced apoptosis in CML cells. On a molecular level, IFNγ-exposed CML cells showed a significantly reduced caspase-3 activation and PARP-1 cleavage as well as an increased expression of anti-apoptotic molecule xIAP. Finally, IFNγ diminished TKI-induced downregulation of Jak-2 and STAT-5 phosphorylation and increased nuclear expression of RUNX-1, which may at least in part contribute to the reduced sensitivity to TKI effects. Our results demonstrate that IFNγ released by activated T or NK cells may interfere with the therapeutic effects of TKI in CML. Our findings may have important implications for the understanding of inflammation-mediated BCR-ABL independent resistance mechanisms.

Modeling Ponatinib Resistance in BCR-ABL1+ Cell Lines: Implications for Ponatinib Resistance in TKI-Resistant and TKI-naïve Patients

Introduction: Tyrosine kinase inhibitor (TKI) resistance remains a major impediment to successful treatment of patients with chronic myeloid leukemia (CML). The 3rd generation TKI ponatinib (Pon), has been demonstrated to successfully overcome BCR-ABL1 kinase domain (KD) mutation based resistance including BCR-ABL1T315I, which inhibits the binding of all other available TKIs. Pon has demonstrated efficacy in CML patients who acquired resistance to other TKIs. However, little is known about the mechanisms which result in resistance to Pon. Here, modes of Pon resistance have been investigated invitro in both TKI naïve and treated BCR-ABL1+ cell lines.Methodology and Results: Pon resistance was generated in 4 BCR-ABL1+ cell lines 1) K562, 2) K562 DOX (increased ABCB1) 3) K562 DOX 55D –resistant to 55 nM dasatinib (Das) and 4) K562 T315I – T315I mutation developed in the presence of Das. Both Das treated cell lines have 3-4 fold higher expression of BCR-ABL1 mRNA compared to control lines. The K562 T315I line contains 44% T315I mutation whereas no KD mutation was found in the other lines. Parental Pon naive controls were maintained in parallel.Resistant cell lines (Table 1) were established by exposing the above cell lines to increasing concentrations of Pon for 660-900 days. The K562 T315IR resistant line demonstrated a >17 fold increase in IC50Pon compared to naïve control. While no expression of ABCB1 or ABCG2 was observed, BCR-ABL1 transcript level was raised 7 fold with a concomitant 4 fold increase in pBcr-Abl by western blot. In addition, the level of T315I in the resistant line increased from 44% to 81%, suggesting that increasing T315I% together with Bcr-Abl upregulation were the major resistant mechanisms in this line.The K562 DOX 55DR line demonstrated transient increases in BCR-ABL1 that reduced to basal levels concomitant with the development of the compound mutations G250E (71%) and E255K (64%). The IC50Pon was increased 47 fold compared to control, and high Das and imatinib (Im) IC50s confirmed the resistance to other TKIs (IC50Das: 1350 nM and IC50Im: 260 μM).In contrast, the Das naïve K562R and K562 DOXR resistant lines did not have demonstrable KD mutations. While 7AAD and annexin V staining confirmed Pon resistance, the IC50Pon was not higher in K562R and only 2 fold higher in K562 DOXR compared to controls, suggesting resistance to Pon was likely mediated by a Bcr-Abl independent mechanism. This was supported by a 2 fold reduction in pBcr-Abl and pSTAT5 in the Pon resistant lines compared to controls. Therefore, the expression levels of alternative pathway molecules were examined to determine possible mechanisms.Axl, a receptor tyrosine kinase has been previously associated with TKI resistance. A 7 fold increase in AXL mRNA was found in K562R line, and the K562 DOXR line demonstrated increased Axl protein. Importantly, after 72 hours incubation with Pon and the Axl inhibitor, R428, the viability of the two resistant lines were significantly reduced (Fig 1), suggesting Axl is likely critical in mediating Pon resistance. Furthermore, increased Axl was associated with increased cell adhesion in both lines, and adherence is a hallmark of Axl expression.Conclusion: As Pon binds to native Bcr-Abl stronger than to Bcr-AblT315I, an increased proportion of T315I reduced sensitivity to Pon in vitro. Pon exposure in a cell line predisposed to KD mutations resulted in the development of the compound mutations G250E and E255K, which rendered the cells insensitive to all tested TKIs. Interestingly the mode of resistance in the two Das naïve lines appears Bcr-Abl independent, and is likely mediated by Axl. While limited to in vitro studies these findings suggest that in the setting of prior TKI exposure, mutations are likely to be a primary form of Pon resistance. In the TKI naïve setting Bcr-Abl independent modes of resistance may be more likely suggesting combination therapeutic approaches may be warranted to overcome or prevent Pon resistance in the high risk setting.Abstract 4515. Table 1:Summary of resistance mechanisms detected in 4 cell lines.Pon culture condition (nM)Pon resistance Bcr-Abl dependent/ independentBCR-ABL1 mRNA(fold ↑ )pBcr-Abl & pSTAT5(fold ↑ )KD mutationIC50Pon(fold ↑ )AXL mRNA(fold ↑ )↑ cell surface AxlAdherenceK562 T315IR100DP74T315I>175✖✖K562 DOX 55DR200DP11G250E E255K47✖✖✖K562R200IDP10.5✖✖7✖✓K562 DOXR200IDP30.5✖2✖✓✓✓= yes; x = no; ↑=increased; DP = dependent; IDP = independent. [Display omitted] [Display omitted] DisclosuresHughes:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding.

Impact Of Baseline (BL) Mutations, Including Low-Level and Compound Mutations, On Ponatinib Response and End Of Treatment (EOT) Mutation Analysis In Patients (Pts) With Chronic Phase Chronic Myeloid Leukemia (CP-CML)

Impact Of Baseline (BL) Mutations, Including Low-Level and Compound Mutations, On Ponatinib Response and End Of Treatment (EOT) Mutation Analysis In Patients (Pts) With Chronic Phase Chronic Myeloid Leukemia (CP-CML)

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

The response to Histone deacetylase inhibitor in Imatinib resistant, BCR-ABL independent, K562 variant with high levels of phosphorylated P38

Abstract Background Imatinib mesylate, a TK inhibitor, is the main treatment for CML but patients may develop drug resistance. Mutations within the Abl kinase domain, increased expression of BCR-ABL or other regulatory mechanisms were suggested to be involved. However, recently the importance of Bcr-Abl independent mechanisms in Imatinib resistance was suggested. Exploring the role of proteins involved in Bcr-Abl independent resistance is of great importance in designing new modalities to overcome resistance or potentiate TKI efficacy.

A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

BackgroundAlthough chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.ResultsThe use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy.ConclusionsThrough the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Abstract 2481Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods:Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results:The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABLL387F). Conclusions:The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures:No relevant conflicts of interest to declare.

The Dark Side of NRF2: Upregulation of NRF2 as a Mechanism for Resistance to Imatinib In CML.

AbstractAbstract 3401Chronic myeloid leukemia (CML) is a myeloproliferative disorder that results from a balanced translocation between chromosomes 9 and 22, creating the Philadelphia chromosome, and giving rise to a 210 kd (p210) fusion protein between Bcr and Abl. In addition to conferring anti-apoptotic properties, proliferative advantage and growth factor independence, BCR-ABL is associated with increased reactive oxygen species (ROS), DNA damage and altered repair that are proposed to play a role in genomic instability and disease progression. Imatinib mesylate, and other tyrosine kinase inhibitors (TKI)s, induce a substantial clinical response in patients with BCR-ABL-positive CML. However, resistance to TKIs through acquisition of mutations in BCR-ABL or BCR-ABL independent mechanisms is an increasing clinical problem. Thus, delineating mechanisms for disease resistance may allow the development of novel therapies for TKI-refractory disease.Cells deal with high levels of intracellular ROS by transcriptionally activating a number of anti-oxidant pathways. In particular, acute increases in ROS levels activate the Nrf2 pathway. Since NRF2 is known to transcriptionally activate drug resistance genes, and some anti-oxidant genes are transcriptionally increased in imatinib-resistant cells, we characterized the role of NRF2 in imatinib resistance in CML. We studied two imatinib-resistant (IR) BCR-ABL-positive cell lines, P210Mo7eIR with no detectable mutations in BCR-ABL and Baf3P210IR with BCR-ABL mutation T315I, derived from isogenic imatinib-sensitive (IS; P210Mo7e and Baf3P210) cells and parental controls (Mo7e and Baf3), and demonstrated that one of the IR cell lines, P210Mo7eIR, had very high steady state levels of Nrf2 (>10-fold) compared with isogenic controls. In addition, real-time PCR studies of NRF2 anti-oxidant target transcripts including, NQO1, NQO2, HMO1, GCLM and GCLC in P210Mo7eIR cells showed that they significantly increased compared with control cells. Importantly, to determine if NRF2 plays a role in the sensitivity of P210Mo7eIR cells to imatinib, we performed siRNA knockdown of NRF2 followed by growth of these cells in the presence of imatinib, and find that downregulation of NRF2 resensitizes P210Mo7eIR cells to imatinib, dramatically increasing cell death, compared with that of siRNA scrambled controls. Furthermore, inhibition of NQO1/2 leads to sensitivity of P210Mo7eIR cells to imatinib. Conversely, overexpression of NRF2 cDNA constructs in imatinib sensitive P210Mo7e renders these cells resistant to imatinib, compared with mutated NRF2 and vehicle controls. To determine whether this mechanism is operative in primary samples from IR cells, we examined 5 IS and 5 IR CML BM samples for steady state levels of NRF2 protein and transcript levels of NRF2 target genes. One of 5 IR CML samples with no BCR-ABL mutations and no IS CML samples had significantly increased NRF2 levels and target anti-oxidant activity. These data suggest that significant upregulation of Nrf2 acute stress pathways may be an alternative mechanism for TKI resistance that does not involves mutations in BCR-ABL. Thus, inhibition of NRF2 itself or its downstream targets may be important in therapeutic strategies in this sub group of TKI resistant CML. Disclosures:No relevant conflicts of interest to declare.

Pretreatment Expression Levels of MDR1 and OCT-1 Predict Response to First Line Imatinib Treatment In Chronic Phase CML Patients

AbstractAbstract 671 Introduction:The goal of imatinib therapy in newly diagnosed CML patients is the rapid achievement of cytogenetic and molecular responses. However, suboptimal response or resistance occur in a significant proportion of patients. BCR-ABL kinase domain mutations and various BCR-ABL independent mechanisms, such as clonal evolution are considered as leading causes of resistance and progression. It has been shown that imatinib efficacy depends on intracellular drug levels which are influenced by the activity of the human organic cation transporter 1 (OCT-1) influx protein and the multidrug resistance 1 (MDR1) efflux transporter protein. We therefore assessed the predictive significance of MDR1 and OCT-1 expression levels for molecular and cytogenetic response of chronic phase CML patients on first line imatinib treatment. Methods:A cohort of 170 newly diagnosed chronic phase CML patients (68 female, median age 53 years, range 19–79) treated with imatinib 400 mg/day in the German CML-Study IV were investigated. Multidrug resistance 1 efflux transporter protein (MDR1) and human organic cation transporter 1 (OCT-1) mRNA expression levels were determined by quantitative reverse transcription PCR using LightCycler™ technology and normalized against beta-glucuronidase (GUS) expression. Cytogenetic response was determined by G-banding metaphase analyses. Cut-off levels were defined by minimizing p-values. The Log-rank test (LR) and Gehan-Breslow-Wilcoxon test (GB) were performed to compare the time to major molecular remission (MMR) and complete cytogenetic remission (CCyR). Results:CCyR was achieved after a median of 6 months (range 3–42), MMR (BCR-ABL levels ≤0.1% on the International Scale) was achieved after a median of 13 months (range 3–43). Further, a significantly higher MMR and CCyR rates were observed in patients with high MDR1 and OCT-1 expression compared to patients with low MDR1 and OCT-1 expression. In addition, a prognostic score resulting in a three group stratification: Good risk, high MDR1 and high OCT-1 expression; intermediate risk, high MDR1 or high OCT-1 expression; poor risk, low MDR1 and low OCT-1 expression was also determined. MMR and CCyR rates were significantly higher in good risk compared to poor risk patients (Table).Months61224Incidence of CCyR (%)Overall285384OCT-1/GUS <0.942754830.0049/0.002OCT-1/GUS >0.94507690MDR1/GUS <1.72852830.07/0.02MDR1/GUS >1.7457291Incidence of MMR (%)Overall103066OCT-1/GUS <0.941025610.036/0.018OCT-1/GUS >0.94114678MDR1/GUS <1.7622610.0047/0.0011MDR1/GUS >1.7164876Risk Group StratificationIncidence of CCyR (%)Poor Risk519580.5/0.20.0001/<0.0001Intermediate Risk163469Good Risk166889Incidence of MMR (%)Poor Risk2550820.089/0.0320.0001/<0.0001Intermediate Risk456286Good Risk7694100 Conclusions:Pretreatment expression levels of MDR1 and OCT-1 appear to predict the achievement of MMR and CCyR under imatinib therapy in chronic phase CML patients over a period of 2 years of therapy. These findings might allow risk stratification in order to tailor the individualized first line therapy in CML. Disclosures:Erben:Novartis: Honoraria, Research Funding. Hochhaus:Novartis, BMS: Consultancy, Honoraria, Research Funding. Mueller:Novartis, BMS: Honoraria, Research Funding.