K562-R As a Model to Study Jak2 in a Non Bcr-Abl Addicted Cell Line

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4412-4412
Author(s):  
Bastianella Perazzona ◽  
Yu-Hsi Lin ◽  
Ralph B. Arlinghaus
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cells ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Janus Kinase ◽  
Cell Fractionation ◽  
Lyn Kinase

Abstract Abstract 4412 Chronic myeloid leukemia (CML) is a hematological disease caused by the fusion protein Bcr-Abl tyrosine kinase. Development of the tyrosine kinase inhibitor Imatinib Mesylate (IM) has significantly improved the long-term survival of early stage CML patients. However, occurrence of drug resistance, permanence of residual disease and recurrence of active leukemia if IM is discontinued, remain problems awaiting solution. Therefore, new therapeutic strategies aimed at targeting alternative signaling pathways or CML progenitor cells that survive IM treatment are needed. We have previously shown that Janus kinase 2 (Jak2) is activated in Bcr-Abl+ cells. We have demonstrated that reduction of Jak2 activity by the Jak2-specific inhibitor TG101209 (TG) or by genetic knock down (Jak2 shRNA and siRNA) in Bcr-Abl+ cell lines, IM-resistant cells and CML blast crisis cell lines resulted in reduced levels of phosphorylation of Tyr177 and of total Bcr-Abl protein. Jak2 inhibition results in diminished activation of the Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5 (Samanta et al., Leukemia 2011). During these studies we observed that K562 cells and IM-resistant cell line K562-R had different susceptibility to the effect of TG, with K562-R showing increase sensitivity to lower concentrations of TG resulting in faster destabilization of the Bcr-Abl protein. Based on these observations, we hypothesized that increased sensitivity of the K562-R cells was due to the different state of activation of Jak2. In addition, based on recent studies by (Dawson et al., Nat 2009) and by (Rinaldi et al., Blood 2010) we also hypothesized that different levels of Jak2 activation may influence the localization of Jak2 in the cell. We used cell fractionation and western blotting analysis to show that in K562-R cells, active Jak2 is mostly localized in the nucleus with a minor pool found in the cytoplasm. In K562 cells, active Jak2 is equally distributed in both cytoplasmic and nuclear compartment. In addition, immuno-fluorescence confocal analysis of total Jak2 distribution in K562 shows a very organized localization of Jak2 at one pole of the cells but this organization is lost in K562-R and total Jak2 appears uniformly distributed within the cell. K562-R cells were isolated as a Bcr-Abl independent IM resistant cell line that expressed high levels of activated Lyn kinase (Donato et al., Blood 2003). We used K562-R as a model to study the role of Jak2 in a non Bcr-Abl addicted cell line. Since we have previously published that Jak2 up-regulates Lyn kinase activity (Samanta et al., Oncogene 2009), we propose that the higher activation of Jak2 in K562-R is the main driver of oncogenicity and IM resistance and that this cell line may be used to model the role of Jak2 in a cell that is not Bcr-Abl “addicted.” Disclosures: No relevant conflicts of interest to declare.

Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3181-3181 ◽  
Author(s):  
Francois-Xavi er Mahon ◽  
Sandrine Hayette ◽  
Valerie Lagarde ◽  
Franck E Nicolini ◽  
Francis Belloc ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Over Expression ◽  
Abl Tyrosine Kinase ◽  
Lyn Kinase

Abstract Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress. In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.

Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2175-2175
Author(s):  
Jean-Max Pasquet ◽  
Romain Gioia ◽  
Claire Drullion ◽  
Valerie Lagarde ◽  
Cedric Leroy ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Tyrosine Kinase Activity ◽  
Tyrosine Residues ◽  
Lyn Kinase ◽  
Resistant Cells

Abstract Abstract 2175 Poster Board II-152 Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl. Disclosures: Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

Evaluation of XIAP as a Molecular Target To Increase Sensitivity to Imatinib.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1381-1381
Author(s):  
Hugo Seca ◽  
Raquel T. Lima ◽  
Jose E. Guimaraes ◽  
M. Helena Vasconcelos
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
K562 Cells ◽  
Viable Cell ◽  
Cell Number ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Viable Cell Number ◽  
Concomitant Treatment ◽  
Xiap Expression

Abstract Imatinib, a potent and selective inhibitor of BCR-ABL, has emerged as an effective drug for targeted therapy of CML. However, resistance to this drug has been arising, mainly due to an overexpression of BCR-ABL, to mutations in the tyrosine-kinase domain of BCR-ABL or to an overexpression of the multidrug resistance protein (Pgp). Cytotoxic drugs induce cellular apoptosis and resistance to these drugs is often due to resistance to apoptosis. Cancer cells may escape apoptosis due to an overexpression of anti-apoptotic proteins such as XIAP, which binds caspases 3, 7 and 9 and inhibits their action. Downregulation of XIAP expression has been documented to increase sensitivity of several cell lines to some conventional drugs. In the current study we investigated if downregulation of XIAP expression, following treatment with XIAP-siRNAs, would increase sensitivity to Imatinib in: i) a sensitive CML cell line (K562), when compared to treatment with control siRNAs and ii) a resistant cell line which overexpresses Pgp (K562Dox), when compared to treatment with Pgp-siRNAs or with control siRNAs. To confirm if the siRNAs were capable of downregulating the expression of their targets, Western Blots were carried out at 24, 48 and 72h after transfection. The cells had been: i) treated with medium only (Blank), ii) transfected with a control-siRNAs, iii) transfected with siRNAs for XIAP and iv) tranfected with siRNAs for Pgp (in the K562Dox cell line only). It was verified that the XIAP-siRNAs decreased XIAP protein levels in both cell lines 48h following transfection and Pgp-siRNAs decreased Pgp protein levels in the resistant cell line (K562Dox) 24h following transfection, when compared to the control-siRNA treatments. To further investigate if the downregulation of XIAP expression (and Pgp in the resistant cell line) caused sensitization of cells to1μM Imatinib, Imatinib was added to the cells previously transfected with siRNAs (for P-gp or XIAP or control siRNAs) and to cells in exponential growth (Blank cells). Viable cell number was counted by the Trypan Blue exclusion assay and apoptosis was verified with the TUNEL assay. Results of viable cell numbers from several experiments indicate that Imatinib treatment on its own, as expected, decreased more the viable cell number of the sensitive cell line than of the resistant cell line (from 100% in the Blank cells to 63 % in the treated K562 cells and to 86 % in the treated K562Dox cells). Furthermore, downregulation of XIAP expression on its own decreased the viable cell number of both cell lines (from 100% in the Blank cells to 62% in the K562 XIAP-siRNA treated cells and to 80% in the K562Dox XIAP-siRNA treated cells). Concomitant treatment with both XIAP-siRNAs and Imatinib further reduced the number of viable cells to 36% in the K562 cells and to 57% in the K562Dox cells. In the resistant cell line, downregulation of Pgp expression on its own reduced the viable cell number from 100% in Blank cells to 84% in the Pgp-siRNA treated cells. Concomitant treatment with Pgp-siRNAs and Imatinib further reduced the viable cell number to 60%. The reduction of viable cell number coincided with an increase in apoptosis. It was concluded that downregulation of XIAP expression may be a good approach to enhance sensitivity to Imatinib, even in the case of existing Pgp overexpression.

Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 746-746
Author(s):  
Kavita B. Kalra ◽  
Xiangfei Cheng ◽  
Marion Womak ◽  
Christopher Gocke ◽  
Jyoti B. Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Breast Cancer Cell Lines ◽  
Hematopoietic Stem

Abstract All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

Evidence for Cooperation of Receptor Tyrosine Kinases and Activating NOTCH Mutations to Hyperactivate mTOR in T-Cell Leukemia: A Rationale Basis for Targeted Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1381-1381
Author(s):  
Adrian Schwarzer ◽  
Johann Meyer ◽  
Martijn Brugman ◽  
Axel Schambach ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Microarray Analysis ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Akt Activation ◽  
Rtk Signaling

Abstract Abstract 1381 T-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. T-ALLs are characterized by recurring chromosomal rearrangements causing aberrant expression of transcription factors (Myb; TAL/SCL; HOX) dividing patients into different subgroups. Activating mutations in NOTCH, the master regulator of T-cell development, are found in more than 60% of T-ALLs independently of subtype. Most T-ALLs display a hyperactivation of the PI3K-AKT-mTOR pathway, a potential target for therapeutic intervention. The master regulator of PI3K-AKT signalling is PTEN, which is frequently inactivated in cancer. Recent data suggests that complete PTEN loss due to mutation is rare in primary human T-ALL, whereas PTEN-inhibiting posttranslational modifications are more common (Barata et al., J. Clin. Invest. 2008, 118). As these modifications decrease, but do not abolish the phosphatase activity of PTEN, we hypothesized that further input from tyrosine kinases, particularly receptor tyrosine kinases (RTK), may be needed to sustain PI3K-AKT-mTOR activation. In order to investigate how RTK-signaling may contribute to the pathogenesis of T-ALL we used an established murine bone marrow transplantation model (Li et al. Blood 2009, 113). To mimic tyrosine-kinase signaling we expressed δTrkA, a constitutively active TRKA receptor tyrosine kinase (TRK =tropomyosin-related kinase) from gammaretroviral or lentiviral vectors in c-kit+ Sca-1+ Lin− (KSL) cells. Intravenous injection of δTrkA-transduced hematopoietic cells in C57BL6 mice (n=10) induced transplantable T-ALL with a latency of about 120 days. The resulting T-ALLs could be propagated in culture as clonal cell lines. Signaling studies showed that δTRKA activates predominantly ERK upon expression in murine hematopoietic cell lines. However, the obtained δTRKA+ T-ALL lines (n=7) showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of AKT-mTOR and absent ERK phosphorylation, resembling human T-ALL. High AKT activation was uniformly detected regardless of PTEN protein expression in all but one T-ALL (#003). To understand the rewired signaling network we looked for a potential contribution of insertional mutagenesis and chromosomal aberrations. Array-CGH showed homozygous deletions on chr14c2 involving the T-cell receptor alpha and delta genes in 3/3 cell lines and heterozygous deletions in Ikzf1 in 2/3 cell lines. Viral integration sites showed no common insertion pattern and no insertion in genes implicated in RTK-signaling. The expression of genes in proximity to viral integrations (±500 kb) appeared unaltered as determined by cDNA-microarray analysis of the T-ALL cell line #483 against wild type CD4+CD8+ thymocytes. Microarray analysis revealed enrichment of Notch1 target genes in the T-ALL cell line #483. Sequencing of Notch1 revealed both, PEST domain mutations and the recently described (Aster et al, Blood 2010, 116) RAG mediated 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein, respectively. The one cell line (#003) which retained the original δTrkA signaling pattern had no Notch mutation and could only be cultured on OP9-Delta-like-1 stroma cells, highlighting the importance of Notch signaling. As this cell line was established from a mouse displaying an enlarged thymus, but no full manifestation of T-ALL, our data suggests that acquisition of Notch mutations is a late, but necessary step required for overt leukemia, whereas the initiating events may arise in kinase signaling pathways of prethymic progenitors. All T-ALL cell lines were sensitive to mTOR or Notch inhibition with Rapamycin or Compound E, respectively. Finally, we used phosphoprotein-arrays to monitor the phosphorylation of 42 RTK in childhood T-ALL samples with different activating NOTCH mutations (n=5) and detected several activated RTK (e.g. MSPR, FGFR, ErbB4, VEGFR) in the patient samples. Taken together, our findings suggest a cooperation of RTK and activating NOTCH mutations in mTOR activation seen in T-ALL and encourage further investigation of 1) aberrant RTK-signaling in T-ALL 2) the role of RTK activation in creating a preleukemic cell clone, 3) evaluation of combined therapy targeting RTKs and NOTCH, and 4) the role of activated NOTCH on mTORC2-AKT activation independently of PTEN. Disclosures: Baum: Patent office: Patents & Royalties.

BGB324 Inhibits BCR-ABL TKI-Resistant Chronic Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1569-1569
Author(s):  
Isabel Ben Batalla ◽  
Robert Erdmann ◽  
Heather Jørgensen ◽  
Rebecca Mitchell ◽  
Thomas Ernst ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Research Funding ◽  
Combined Treatment ◽  
K562 Cells ◽  
Resistant Cell ◽  
Additive Effect ◽  
Significant Prolongation

Abstract Resistance and CML stem cell persistence preclude cure for the majority of patients treated with tyrosine kinase inhibitor (TKI) therapies. We demonstrated that the receptor tyrosine kinase (RTK) Axl of the Tyro-3, Axl, Mer (TAM) family is expressed by TKI-sensitive and -resistant CML cells (Erdmann R. et al. ASH 2013 and 2014). We have shown that blockade of the Gas6-Axl axis by the small molecule Axl inhibitor BGB324 (BerGenBio) represents a therapeutic target in AML. We are currently investigating BGB324 in a Phase 1b trial in refractory AML patients and in those non-eligible for intensive chemotherapy (BGBC003, NCT02488408). We hypothesised that Axl represents a tractable therapeutic target even in the most resistant forms of CML. Upon treatment with imatinib KCL-22 and K562 cells showed upregulation of Axl at the protein level indicating that Axl might be involved in resistance towards TKIs in CML. Consistently, Axl levels were higher in MNCs of TKI-resistant patients compared to -sensitive patients after 6 months of treatment (n=17/20, 1±0.4 vs. 0.16±0.03; *p<0.05). Upon combined treatment of KCL-22 and K562 cells with BGB324 and imatinib we detected an additive effect of growth inhibition (KCL-22 cells; n=3, viability 66.0±0.5% BGB324, 52.0±1.1% imatinib, 42.3±1.5% combo; combo vs. IM *p<0.01 and combo vs. BGB324 *(p<0.0001), and not shown). Analysis of intracellular signal transduction in these cell lines indicated that Axl induces phosphorylation of Stat5 by BCR-ABL independent pathways because we detected an additive effect of inhibition of Stat5 phosphorylation when combining imatinib and BGB324. We could not detect an additive inhibitory effect on phosphorylation of Erk and Akt. Consistently, combined BCR-ABL and Axl blockade by means of imatinib and shRNA respectively, demonstrated an additive effect in reducing cell viability in KCL-22 and K562 cells (KCL-22 cells; n=3, viability 84.5±0.8% shControl+imatinib, 74.5±2.6% shAxl, 50.4±0.9% shAxl+imatinib; shAxl+IM vs. IM *(p<0.0001) and vs. shAxl *(p<0.001) and not shown). We next investigated Axl activation in TKI insensitive BCR-ABL+ cell lines. In addition we tested a novel Ponatinib-resistant cell line KCL-22 PonR generated by subcloning parental KCL-22 in increasing concentrations of ponatinib. BCR-ABL is unmutated in these cells; oncoprotein kinase activity is switched off but cell death is not induced with 2mM ponatinib. We found that Axl phosphorylation was higher in the TKI-resistant cell lines BaF3/T315I, KCL-22 T315I and KCL-22 PonR when compared to the parental cell lines (n=3, 139±3.8% KCL-22 T315I, 214±1.3% KCL-22 PonR with respect to KCL-22 WT, *p<0.001 for both comparisons; 169±8.7% with respect to BaF3/p210, *p<0.005). Treatment with BGB324 inhibited cell proliferation with an IC50of 726, 3178 and 2720nM for BaF3/T315I, KCL-22 T315I and KCL-22 PonR, respectively. BGB324 could induce apoptosis and reduce proliferation in these cell lines. Furthermore, BGB324 blocked growth of colonies and induced apoptosis of T315I-mutated and pan-TKI-resistant (including ponatinib) primary CML MNCs. The finding that BGB324 inhibits TKI-resistant CML was further corroborated with KCL-22 T315I mutated and KCL-22 PonR xenograft models. In both models we observed a significant tumor growth reduction upon treatment with 25 mg/kg BGB324 twice daily compared to placebo leading to a 34% and 58% reduction in tumor volume, p=0.0044, p=0.0021 for KCL-22 T315I and KCL-22 PonR, respectively). Cell proliferation was quantified by pHH3 analysis indicating a significant reduction in KCL-22 T315I and KCL-22 PonR tumors (n=8, 242.8±10.9 vs. 182.2±8.1, *p<0.0001; n=9, 259.5±9.3 vs. 213.2±6.8; *p<0.0001, respectively). Furthermore, we observed a significant decrease of Axl, Erk and Stat5 phosphorylation after treatment with BGB324 for 8 days. We also investigated the therapeutic effect of BGB324 in a systemic model, by transplantation of KCL-22 PonR into sublethally irradiated NSG mice. In this model, treatment with 25 mg/kg BGB324 twice daily resulted in significant prolongation of overall survival (median OS 36 days (control) vs 43 days (BGB324), n=5 *p<0.05). In summary, our data highlight the advantage to be gained from inhibition of Axl even in the most resistant CML cells, and support the need for human clinical trials of the novel inhibitor BGB324 alone and in combination with TKIs. Disclosures Schafhausen: Novartis: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hochhaus:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Holyoake:Novartis: Research Funding; BMS: Research Funding. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding.

scholarly journals Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2668-2668
Author(s):  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Joseph Ahmann ◽  
Cecilia Bonolo De Campos ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Sensitive Cell ◽  
Advisory Committees ◽  
Bcl2 Family ◽  
Family Regulation

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Arsenic trioxide resistance in acute promyelocytic leukemia: More to it than PML mutations

2020 ◽  
Author(s):  
Nithya Balasundaram ◽  
Saravanan Ganesan ◽  
Ezhilarasi Chendamarai ◽  
Hamenth Kumar Palani ◽  
Arvind Venkatraman ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Resistant Cell Line ◽  
Promyelocytic Leukemia ◽  
Genetic Mutations ◽  
List Type

AbstractAcquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further.Key pointsMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation.Combination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients.

Molecular mechanism(s) of resistance to vandetanib in medullary thyroid carcinoma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15628-e15628
Author(s):  
Brittany Glassberg ◽  
Sophia Khan ◽  
Alex Pemov ◽  
Robert Hawley ◽  
Brigitte C. Widemann ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Rna Sequencing ◽  
Medullary Thyroid Carcinoma ◽  
Cell Lines ◽  
Resistant Cell ◽  
Knock Out ◽  
Medullary Thyroid ◽  
Genome Wide

e15628 Background: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid. Pediatric cases of this cancer are associated with the diagnosis of multiple endocrine neoplasia, which is caused by a mutation in the rearranged during transfection ( RET) gene. Vandetanib, an oral receptor tyrosine kinase inhibitor, is approved for the treatment of patients with progressive MTC. While there is a response rate of 50%, the majority of patients eventually develop resistant disease. The goal of this work is to understand genetic and epigenetic underpinnings of sensitivity and resistance to vandetanib, and develop novel synergistic combination therapies in medullary thyroid carcinoma. Methods: The TT cell line (RET mutation p.C634W) was cultured in increasing concentrations of vandetanib in order to generate a vandetanib resistant cell line. Both vandetanib-sensitive and vandetanib-resistant lines were evaluated. Each line underwent exome sequencing, RNA sequencing, and methylation array analysis. In parallel we performed Genome-wide CRISPR knock-out and CRISPR activation of both cell lines using the TKO Version 3 Library, consisting of 71,090 gRNAs targeting 18,000 genes, and the Calabrese P65-HSF activation Library, using 56,762 guides for 18,000 genes. Results: Both whole exome and RNA sequencing demonstrate increased expression of RET C634W in both cell lines, to a significantly greater extent in the drug-resistant line than in the sensitive line. RNA sequencing demonstrates differential expression of transcripts between the vandetanib-sensitive and vandetanib-resistant cell lines, including multidrug-resistance 1 (which confers drug resistance in other cancers) and autotaxin (promotes cell survival). Genome-wide CRISPR knock-out showed enrichment of genes necessary for growth suppression by vandetanib, including MACROD2, GORASP2, and MAP3K genes. Gene set enrichment analysis of the CRISPR knock-out data showed enrichment of the proteasome pathway as a potential candidate of growth suppression by vandetanib, which was validated via exposure of the cell lines to bortezomib (IC50 = 3.62nM), a molecularly targeted proteasome-inhibitor. Conclusions: DNA mutations and epigenetic modification confer resistance of medullary thyroid carcinoma to tyrosine kinase inhibition. Adding further therapeutic agents to target these genetic alterations is a potential strategy for overcoming resistance.

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