Dasatinib Resistance Research Articles

Functional Characterization of Transforming Growth Factor-β Signaling in Dasatinib Resistance and Pre-BCR+ Acute Lymphoblastic Leukemia.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

The acquisition order of leukemic drug resistance mutations is directed by the selective fitness associated with each resistance mechanism

In Chronic Myeloid Leukemia, the transition from drug sensitive to drug resistant disease is poorly understood. Here, we used exploratory sequencing of gene transcripts to determine the mechanisms of drug resistance in a dasatinib resistant cell line model. Importantly, cell samples were collected sequentially during drug exposure and dose escalation, revealing several resistance mechanisms which fluctuated over time. BCR::ABL1 overexpression, BCR::ABL1 kinase domain mutation, and overexpression of the small molecule transporter ABCG2, were identified as dasatinib resistance mechanisms. The acquisition of mutations followed an order corresponding with the increase in selective fitness associated with each resistance mechanism. Additionally, it was demonstrated that ABCG2 overexpression confers partial ponatinib resistance. The results of this study have broad applicability and help direct effective therapeutic drug usage and dosing regimens and may be useful for clinicians to select the most efficacious therapy at the most beneficial time.

Efficacy and Safety of Flumatinib in Treatment of Patients with Chronic Myeloid Leukemia

To analyze the efficacy and safety of flumatinib in the treatment of patients with chronic myeloid leukemia (CML). The clinical data of 56 CML patients treated with flumatinib from January 2020 to December 2021 in the First Affiliated Hospital of Nanchang University were retrospectively analyzed. Patients were divided into three groups: 35 new diagnosed CML patients treated with flumatinib (group A), 10 patients with imatinib/dasatinib intolerance (group B) and 11 patients with imatinib/dasatinib resistance (group C) switched to flumatinib treatment, respectively. The molecular response and adverse effects of flumatinib treatment were evaluated. In group A, the early molecular response (EMR) at 3 months was 40.0%, and the major molecular response (MMR) at 6 and 12 months was 43.7% and 46.2%, respectively. In group B, the EMR was 50.0% at 3 months, and the MMR was 70.0% and 66.2% at 6 and 12 months, respectively. Among evaluable patients, 6 cases in group B achieved molecular response of 4.5 (MR4.5) at 12 months after switching to flumatinib treatment. In group C, 3 cases who switched from imatinib resistance to flumatinib achieved MR4.5 at 12 months, but 2 cases who switched from dasatinib resistance to flumatinib failed. Subgroup analysis showed significant differences in EUTOS long-term survival (ELTS) scores for patients in the medium-risk/high-risk group compared with those in the low-risk group for 3-month EMR (18.8% vs 57.9%), 6-month MMR (15.4% vs 63.2%) and 12-month MR4.5 (15.4% vs 69.2%) (P =0.036, P =0.012,P =0.015). The most common adverse effect in group A was thrombocytopenia, accounting for 54.5%, and 22.8% (8/35) patients discontinued the drug due to haematological adverse effects. Compared with patients who did not discontinue the drug or whose recovery time from discontinuation due to haematological toxicity was <1 month, patients whose recovery time from discontinuation was ≥1 month had a significantly worse 3-month EMR, 6-month MMR and 12-month MR4.5 (P =0.028, P =0.021, P =0.002). Flumatinib has better molecular response and tolerance in patients with primary, imatinib/dasatinib-intolerant or resistant CML. Medium-risk/high-risk in ELTS score and time to recovery from discontinuation due to haematological toxicity ≥1 month are important factors influencing achievement of better molecular response in flumatinib treatment.

Abstract 1681: The alternative mechanism of Src inhibition in colon cancer cells

Abstract In colon cancer, Src showed the high expression in tumor cells and was known as an important migration marker that plays the role in coordinate with many important cellular signal pathways. According to previous studies, cell viability and migration ability can be inhibited by Src inhibitor, Dasatinib. However, the clinical study demonstrated the survival rate has no significant increase in Dasatinib treatment. Therefore, we want to figure out what is the real function of Src in colon cancer cell in this study. HT-29, colon cancer cell with epithelial cell morphology was used in the study. After treated with Dasatinib, cells were followed by reattachment experiment. Then the cell growth, migration ability, signaling pathway transition, molecular localization and expressions were analyzed. The results showed Src inhibition can reduce EGFR activation on cell-cell contacts and increased Akt phosphorylation upon EGF stimulation. We further found Dasatinib has limited effect on cell toxicity, however, can be the selector to separate two cell populations, Dasatinib sensitive (DSTS) and Dasatinib resistance (DSTR) cells, interestingly, the DSTS cells were grown as colony phenotype efficiently and majorly through EGFR/AKT pathway. Meanwhile, DSTR was demonstrated the promotion of collective cell migration ability by EGFR/Ecad pathway. The results of this study suggested Src activity plays novel role in switch EGFR/Akt and EGFR/Ecad pathways in colon cancer cells and harboring different strategies to survive, therefore, the alternative resistance mechanism of Src inhibitor in colon cancer cells may need be concerned in clinical application. Citation Format: Yi-Wen Lu, Xiang-Ling Hou, Yen-Tse Lu, Chia-Wen Lin, Chien-Chi Huang, Wei-Ting Chao. The alternative mechanism of Src inhibition in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1681.

Activation of ERK1/2 by MOS and TPL2 leads to dasatinib resistance in chronic myeloid leukaemia cells.

The development of BCR::ABL1 tyrosine kinase inhibitors (TKIs), such as dasatinib, has dramatically improved survival in cases of chronic myeloid leukaemia (CML). However, the development of resistance to BCR::ABL1 TKIs is a clinical problem. BCR::ABL1 TKI resistance is known to have BCR::ABL1-dependent or BCR::ABL1-independent mechanisms, but the mechanism of BCR::ABL1 independence is not well understood. In the present study, we investigated the mechanism of BCR::ABL1-independent dasatinib resistance. The expression and activation level of genes or proteins were evaluated using array CGH, real time PCR, or western blot analysis. Gene expression was modulated using siRNA-mediated knockdown. Cell survival was assessed by using trypan blue dye method. We found that dasatinib-resistant K562/DR and KU812/DR cells did not harbour a BCR::ABL1 mutation but had elevated expression and/or activation of MOS, TPL2 and ERK1/2. In addition, MOS siRNA, TPL2 siRNA and trametinib resensitized dasatinib-resistant cells to dasatinib. Moreover, expression levels of MOS in dasatinib non-responder patients with CML were higher than those in dasatinib responders, and the expression of TPL2 tended to increase in dasatinib non-responder patients compared with that in responder patients. Our results indicate that activation of ERK1/2 by elevated MOS and TPL2 expression is involved in dasatinib resistance, and inhibition of these proteins overcomes dasatinib resistance. Therefore, MOS, TPL2 and ERK1/2 inhibitors may be therapeutically useful for treating BCR::ABL1-independent dasatinib-resistant CML.

Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.

MicroRNA-like snoRNA-Derived RNAs (sdRNAs) Promote Castration-Resistant Prostate Cancer.

We have identified 38 specifically excised, differentially expressed snoRNA fragments (sdRNAs) in TCGA prostate cancer (PCa) patient samples as compared to normal prostate controls. SnoRNA-derived fragments sdRNA-D19b and -A24 emerged among the most differentially expressed and were selected for further experimentation. We found that the overexpression of either sdRNA significantly increased PC3 (a well-established model of castration-resistant prostate cancer (CRPC)) cell proliferation, and that sdRNA-D19b overexpression also markedly increased the rate of PC3 cell migration. In addition, both sdRNAs provided drug-specific resistances with sdRNA-D19b levels correlating with paclitaxel resistance and sdRNA-24A conferring dasatinib resistance. In silico and in vitro analyses revealed that two established PCa tumor suppressor genes, CD44 and CDK12, represent targets for sdRNA-D19b and sdRNA-A24, respectively. This outlines a biologically coherent mechanism by which sdRNAs downregulate tumor suppressors in AR-PCa to enhance proliferative and metastatic capabilities and to encourage chemotherapeutic resistance. Aggressive proliferation, rampant metastasis, and recalcitrance to chemotherapy are core characteristics of CRPC that synergize to produce a pathology that ranks second in cancer-related deaths for men. This study defines sdRNA-D19b and -A24 as contributors to AR-PCa, potentially providing novel biomarkers and therapeutic targets of use in PCa clinical intervention.

Gene Network Analysis of Hepatocellular Carcinoma Identifies Modules Associated with Disease Progression, Survival, and Chemo Drug Resistance.

BackgroundHepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. HCC transcriptome has been extensively studied; however, the progress in disease mechanisms, prognosis, and treatment is still slow.MethodsA rank-based module-centric workflow was introduced to analyze important modules associated with HCC development, prognosis, and drug resistance. The currently largest HCC cell line RNA-Seq dataset from the LIMORE database was used to construct the reference modules by weighted gene co-expression network analysis.ResultsThirteen reference modules were identified with validated reproducibility. These modules were all associated with specific biological functions. Differentially expressed module analysis revealed the crucial modules during HCC development. Modules and hub genes are indicative of patient survival. Modules can differentiate patients in different HCC stages. Furthermore, drug resistance was revealed by drug-module association analysis. Based on differentially expressed modules and hub genes, six candidate drugs were screened. The hub genes of those modules merit further investigation.ConclusionWe proposed a reference module-based analysis of the HCC transcriptome. The identified modules are associated with HCC development, survival, and drug resistance. M3 and M6 may play important roles during HCV to HCC development. M1, M3, M5, and M7 are associated with HCC survival. High M4, high M9, low M1, and low M3 may be associated with dasatinib, doxorubicin, CD532, and simvastatin resistance. Our analysis provides useful information for HCC diagnosis and treatment.

P04.04 Optimizing dasatinib for glioblastoma treatment

Abstract BACKGROUND Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis, even with surgical and chemoradiotherapy. Expression profiling studies classify IDH-wildtype Glioblastoma into three subtypes: Proneural (PN), mesenchymal (MES) and classical (CL). A promising target to inhibit in Glioblastoma is the non-receptor tyrosine kinase and proto-oncogene SRC. After robust pre-clinical results, SRC inhibitors like dasatinib did not improve survival of Glioblastoma patients after recurrence in clinical trials. MATERIAL AND METHODS Consolidating efforts to personalize cancer therapy, we use in silico analyses backed by in vitro and in vivo experiments on Glioblastoma stem-like cells (GSCs) derived from primary patient tumors to present a novel stratification strategy for dasatinib therapy in glioblastoma. To further tackle dasatinib resistance in GSCs, a pooled shRNA library against 5000 genes was combined with dasatinib to identify genes whose knockdown sensitizes GSCs to dasatinib. This was integrated with proteomics and phosphoproteomics data of dasatinib inhibited GSCs. RESULTS We found MES tumors with high expression of SERPINH1 to be sensitive to dasatinib inhibition, compared to the CL and PN subtypes. Interestingly, SRC phosphorylation status did not predict the efficacy of dasatinib inhibition. Computational analyses integrating data from the loss-of-function dropout viability screen and proteomics/phosphoproteomics using a novel modification of the SamNet algorithm identified Wee1, a tyrosine kinase involved in cell-cycle signaling, as a potential combination inhibition target with dasatinib. Further validation experiments showed a robust synergistic effect through combination of dasatinib and the wee1 inhibitor, MK-1775 in PN GSCs. CONCLUSION This study highlights strategies to optimize dasatinib treatment in different glioblastoma subtypes. While the stratification of patients harboring mesenchymal glioblastoma with SERPINH1 overexpression could provide an option in this particular subtype, combining dasatinib or other SRC inhibitors with Wee1 inhibitors could present an additional possibility for treating resistant proneural tumors

MEK inhibition overcomes resistance to EphA2-targeted therapy in uterine cancer

BackgroundOur pilot clinical study of EphA2 inhibitor (dasatinib) plus paclitaxel and carboplatin showed interesting clinical activity in endometrial cancer with manageable toxicity. However, the underlying mechanisms of dasatinib resistance in uterine cancer are unknown. Here, we investigated potential mechanisms underlying resistance to EphA2 inhibitors in uterine cancer and examined the anti-tumor activity of EphA2 inhibitors alone and in combination with a MEK inhibitor. MethodsWe evaluated the antitumor activity of EphA2 inhibitors plus a MEK inhibitor using in vitro and in vivo orthotopic models of uterine cancer. ResultsEphA2 inhibitor induced MAPK in dasatinib-resistant uterine cancer cells (HEC-1A and Ishikawa) and BRAF/CRAF heterodimerization in HEC-1A cells. EphA2 inhibitor and trametinib significantly increased apoptosis in cancer cells resistant to EphA2 inhibitors compared with controls (p < 0.01). An in vivo study with the orthotopic HEC-1A model showed significantly greater antitumor response to combination treatment compared with dasatinib alone (p < 0.01). Combination treatment increased EphrinA1 and BIM along with decreased pMAPK, Jagged 1, and c-MYC expression in dasatinib-resistant cells. In addition, Spearman analysis using the TCGA data revealed that upregulation of EphA2 was significantly correlated with JAG1, MYC, NOTCH1, NOTCH3 and HES1 expression (p < 0.001, r = 0.25–0.43). Specifically, MAP3K15 and the NOTCH family genes were significantly related to poor clinical outcome in patients with uterine cancer. ConclusionsThese findings indicate that the MAPK pathway is activated in dasatinib-resistant uterine cancer cells and that EphrinA1-mediated MEK inhibition overcomes dasatinib resistance. Dual targeting of both EphA2 and MEK, combined with chemotherapy, should be considered for future clinical development.

Selected Articles from This Issue

Ewing sarcoma is largely driven by pathognomonic gene fusions involving EWSR1, particularly the EWS-FLI1 fusion, which are chiefly responsible for driving the transcriptional and metabolic rewiring associated with these cancers. However, previous work has also uncovered an oncogenic role for the scaffold protein menin in metabolic rewiring of Ewing sarcoma and other tumors, though the mechanism underlying this role is undefined. In this study, Jiménez and colleagues show that EWS-FLI1 and menin serve as upstream regulators of ATF4, a master regulator of the serine biosynthesis pathway. EWS-FLI1 directly binds the ATF4 promoter and both EWS-FLI1 and menin are integral for the induction of serine synthesis pathway downstream of ATF4, as inhibition of either gene resulted in loss of ATF4 expression and reduced activation of the serine biosynthesis gene program. Interestingly, menin inhibition also caused reduced ATF4 activity in B-cell acute lymphoblastic leukemia bearing MLL rearrangement, suggesting a broader role for menin in metabolic regulation. Taken together, the data uncover new mechanisms underlying the metabolic reprogramming observed in Ewing sarcoma and elucidate a pro-tumorigenic metabolic role for menin that may span a variety of different cancers.Prostate cancer is clinically managed by ablation of circulating testosterone and/or blockade of androgen receptor (AR) signaling, but invariably tumors progress to a castrate-resistant (CRPC) state. Numerous molecular drivers of castration resistance have been described, including hyperactivation of NF-κB signaling. However, strategies to overcome this resistance mechanism have proven elusive due to poor oral bioavailability of NF-κB pathway inhibitors. Here, Morel and colleagues demonstrate effective targeting of the NF-κB axis in CRPC in vivo using a novel orally bioavailable small molecule, dimethylaminoparthlide (DMAPT). Castration of mice bearing prostate tumors caused an increase in NF-κB signaling, leading to increased expression of AR and AR splice variants associated with a CRPC state. However, combination of castration with DMAPT prevented the induction of constitutively active AR variants. The authors show that this effect was specifically reliant on the effective inhibition of phosphorylated p65 NF-κB activity. Taken together, the data support the development of DMAPT as a therapeutic strategy to extend the efficacy of AR-targeted therapy by preventing or delaying the onset of castration resistance.Desmoplastic small round cell tumors (DSRCT) are rare, poorly differentiated cancers that are thought to arise from the peritoneum and which are marked by a pathognomonic EWSR1-WT1 gene fusion. The rarity of this tumor typ. has thus far precluded randomized trials and few laboratory models are available, highlighting an urgent need for additional insights and potential therapeutic targets. Here, Slotkin, Bowman, and their colleagues report genomic and transcriptomic data from a cohort of 78 DSRCT patients as well as a new bank of patient-derived xenografts for the study of DSRCT biology. The authors found that, aside from the pathognomonic fusion, DSRCT has a relatively stable genome with few recurrent mutations. Notably, however, FGFR4 was found to be highly expressed in DSRCT overall, with additional cases bearing FGFR4 amplification and/or activating mutations. These data provide new insights into the biology of DSRCT, including the role of FGFR4 as a potential therapeutic target, and present the first available bank of DSCRT laboratory models to the research community.c-Kit is a frequently activated proto-oncogene in acral and mucosal melanoma, but the c-Kit inhibitor dasatinib has limited efficacy in the clinic. In this study, Sabbah and colleagues perform pathway analysis to elucidate mechanisms of dasatinib resistance in melanoma. The authors report that dasatinib treatment achieves the goal of silencing the MAPK and PI3K/Akt pathways downstream of c-Kit, indicating an on-target effect. However, at pharmacological doses of dasatinib, the authors also observed activation of CREB transcriptional activity leading to induction of MITF and Bcl-2 expression via the CRTC3 pathway. Indeed, forced expression of MITF prevented any response to dasatinib in c-Kit mutant/amplified melanoma models, but inhibition of Bcl-2 using pharmacological agents restored sensitivity to dasatinib. Taken together, these data suggest that dasatinib treatment stimulates its own mechanism of resistance via induction of the pro-survival protein Bcl-2 and may also explain the modest benefits observed in dasatinib clinical trials.

Repurposing Dasatinib for Ibrutinib-Resistant Diffuse Large B-Cell Lymphoma

Diffuse Large B-Cell Lymphoma (DLBCL) is the most frequent B-cell Non-Hodgkin Lymphoma (B-NHL). It derives from germinal-center (GC) B cells, and has been classified into GC B cell like (GCB) and activated B cell like (ABC) subtypes, which differ in their cell of origin, genetic alterations and responses to therapy, with ABC having inferior prognosis in response to the current immuno-chemotherapeutic regimen (R-CHOP). This standard first-line treatment is successful in ~70% of cases based on 5-year overall survival, but the remaining ~30% of patients remains incurable. While recently developed drugs, including the Bruton Tyrosine Kinase (BTK) inhibitor Ibrutinib and PI3K-delta inhibitors, are providing novel targeted strategies, their impact on DLBCL is still limited to a fraction of cases, mostly in the ABC subtype. In order to identify drugs that can be repositioned for selective efficacy against DLBCL subtypes (GCB- and ABC-DLBCL) compared to non-GC derived lymphomas, we screened a focused set including 2,160 FDA-approved drugs and other targeted drugs in advanced clinical testing on a panel of 9 cell lines consisting of 6 DLBCL (3 ABC and 3 GCB) and 3 Mantle Cell Lymphoma (MCL) lines. This screen identified the multi-kinase inhibitor Dasatinib as the most DLBCL-specific agent. Dose-response experiments on an extended panel of 32 DLBCL lines showed that Dasatinib has broader activity than Ibrutinib, since it is active against all ABC-DLBCL Ibrutinib-sensitive lines, as well as an additional set of 9 ABC- and GCB-DLBCL lines. Notably, Dasatinib overcomes Ibrutinib-resistance caused by BTK C481S mutation, as shown by the fact that Dasatinib is active against 3 Ibrutinib-sensitive ABC-DLBCL lines rendered Ibrutinib-resistant by transduction of BTK C481S mutant. In these cell lines, Dasatinib, analogous to Ibrutinib, is unable to suppress BTK-C481S auto-phosphorylation at Tyrosine 223, indicating that it may act independently of BTK. To understand which kinase or combination of kinases needs to be suppressed for Dasatinib activity, we employed Dasatinib-resistant, gatekeeper mutants of each BCR-associated Src-Family Kinase (LYN, FYN and BLK), which can all be targeted by Dasatinib, alone or in combination. Our results showed that FYN suppression is essential for Dasatinib activity in both DLBCL subtypes. Integrated genomic and bioinformatics approaches to explore genetic determinants of resistance to Dasatinib revealed PTEN disruption as the most significant predictor. PTEN is inactivated in the majority of resistant lines by both genetic and epigenetic mechanisms, resulting in the inability by Dasatinib to restrict PI3K-mediated AKT activation. These data were further validated by showing that 3 Dasatinib-sensitive DLBCL lines were made resistant by transduction of a constitutively active PIK3CA mutant (PIK3CA-H1047R). Notably, suppression of AKT activation by mTORC2 inhibitors can revert Dasatinib resistance, suggesting that combinatorial strategies involving Dasatinib and drugs targeting the PI3K/AKT/mTOR axis may be effective in PTEN-negative DLBCLs. The above results were validated in vivo using DLBCL xenograft mouse models. Overall, these results provide a preclinical framework for Dasatinib-based targeted therapies for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Systematic analysis reveals a lncRNA-miRNA-mRNA network associated with dasatinib resistance in chronic myeloid leukemia.

Chronic myelogenous leukemia (CML) is a malignant tumor formed by the clonal proliferation of bone marrow hematopoietic stem cells. CML is a relatively rare disease, mainly affecting elderly patients, but the prevalence of CML is expected to increase dramatically. The tyrosine kinase inhibitors (TKIs) have changed the CML patients' treatment patterns and improved its treatment effect, but drug resistance still remains a significant problem to be solved. Therefore, the identification of biomarkers of CML resistance involved therein is essential for treatment and prognosis prediction. Bioinformatics was used to analyze and construct a lncRNA-miRNA-mRNA network of CML resistance to dasatinib and predict key lncRNAs. By screening differentially expressed genes in CML resistant to dasatinib and comprehensively analyzing their functions and signal pathways, the core genes in these differential genes were found, and by predictive analysis of the upstream targets of these core genes. Finally, a network diagram containing lncRNA, miRNA, and mRNA was constructed. MALAT1 as a lncRNA may be a tumor suppressor in patients with CML. According to our data, MALAT1 may have potential role as a molecular biomarker for the occurrence and development of CML resistance to dasatinib.

Inhibition of Akt/mTOR pathway overcomes intrinsic resistance to dasatinib in triple-negative breast cancer

Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.

Targeting EGFR exon 20 insertions in non-small cell lung cancer by exploiting a dependency on parallel SRC signalling

Abstract Background Inframe insertions in exon 20 of the epidermal growth factor receptor (EGFR) gene are oncogenic drivers in non-small cell lung cancer (NSCLC). However, unlike more common EGFR mutations, low frequency EGFR exon 20 insertions are resistant to clinically approved EGFR inhibitors and are associated with poor patient prognosis. There is an unmet clinical need to identify novel therapies that will be effective for patients with EGFR exon 20 insertions. Methods Cell viability of three patient-derived NSCLC cell line models harbouring EGFR exon 20 insertions, CUTO14 (A767_V769dupASV), CUTO17 (N771_H773dupNPH) and CUTO18 (S768_D770dupSVD) was assessed after treatment with a chemical compound screen targeting cancer-associated pathways. Western blotting was used to probe cell signalling following dasatinib treatment. Mutant SRC expression constructs were introduced into cell lines via lentiviral transduction and depletion of endogenous SRC levels was achieved using RNAi. Models of acquired drug resistance were developed by long-term drug exposure. Results Out of 58 screened compounds, the tyrosine kinase inhibitor dasatinib was a shared hit across all EGFR exon 20 insertion models and was superior to the EGFR inhibitor gefitinib in cell viability and apoptosis assays. Dasatinib sensitivity was rescued by expression of a gatekeeper mutant SRC that does not bind dasatinib, whilst all models were sensitive to RNAi-mediated depletion of endogenous SRC expression. Importantly, models of acquired dasatinib resistance maintain sensitivity to poziotinib, an EGFR inhibitor that can target EGFR exon 20 insertions. Similarly, dasatinib effectively inhibits cell viability of a model of acquired resistance to poziotinib, indicating two independent, targetable signalling nodes. Combined treatment with dasatinib and poziotinib prevented the emergence of drug resistance. Conclusions EGFR exon 20 insertion lung cancer models are sensitive to inhibition of parallel SRC signalling via dasatinib treatment. In a patient population with limited therapeutic options, an upfront rational combination of dasatinib with EGFR inhibitors that target EGFR exon 20 insertions has the potential to achieve durable responses. Legal entity responsible for the study Paul Huang. Funding Institute of Cancer Research, Cancer Research UK. Disclosure R. Doebele: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses, Licensing / Royalties: Genentech/Roche; Advisory / Consultancy, Travel / Accommodation / Expenses, Licensing / Royalties: Ignyta; Licensing / Royalties: Foundation Medicine; Advisory / Consultancy, Licensing / Royalties: Loxo Oncology; Advisory / Consultancy: Bayer; Advisory / Consultancy, Shareholder / Stockholder / Stock options, Licensing / Royalties: Rain Therapeutics; Honoraria (self), Advisory / Consultancy: Takeda/Millenium; Licensing / Royalties: Abbot Molecular. All other authors have declared no conflicts of interest.

Identification of biomarkers and their functions in dasatinib-resistant pancreatic cancer using bioinformatics analysis.

Dasatinib is a tyrosine kinase inhibitor, which inhibits tumor proliferation by blocking SRC pathways and is considered as a potential treatment of various epithelial neoplasms, including pancreatic cancer. However, dasatinib efficacy is largely limited due to drug resistance. In the present study, bioinformatics strategies were used to investigate the potential mechanisms of dasatinib-resistance in pancreatic cancer. The gene expression profiles of the Panc0403, Panc0504, Panc1005 (dasatinib-sensitive), SU8686, MiaPaCa2 and Panc1 (acquired dasatinib-resistant) cell lines were obtained from the gene expression omnibus database. The differentially expressed genes (DEGs) were then selected using R software. In addition, gene ontology (GO) and pathway enrichment analysis were performed through the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network was constructed and analyzed to determine the hub genes using the Search Tool for the Retrieval of Interacting Genes database. A total of 472 DEGs, including vimentin, transmembrane 4 l six family member 18 and S100 calcium binding protein P, were identified. Enrichment analysis by GO function demonstrated that DEGs were associated with extracellular components, signal regulation and binding factors. The analysis of the Kyoto Encyclopedia of Genes and Genomes demonstrated that several adenocarcinoma pathways were enriched, including the phosphoinositide 3-kinases/protein kinase B and mitogen-activated protein kinase signaling pathways. Some hub genes were highlighted following the PPI network construction, including Rac family small GTPase 1, laminin subunit α3, integrin subunit β4, integrin subunit α2, collagen type VI α1 chain, collagen type I α2 chain, arrestin β1 and synaptotagmin 1, which may be associated with pancreatic adenocarcinoma prognosis. A total of five out of eight hub genes were highly associated with the overall survival rate (P<0.05). In conclusion, the present study reported novel insights into the mechanisms of dasatinib resistance. Identification of these hub genes may be considered as potential novel treatment targets for dasatinib-resistance in pancreatic cancer.

Dasatinib Treatment Increases Sensitivity to c-Met Inhibition in Triple-Negative Breast Cancer Cells.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib; however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 μM compared to 0.04 ± 0.001 µM in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 ± 0.5 µM compared to an IC50 of 6.8 ± 0.2 µM in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

Dasatinib as an investigational drug for the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia in adults

ABSTRACTIntroduction: Acute lymphoblastic leukemia (ALL) with BCR-ABL1 translocation is an aggressive malignancy that is usually treated with intensive chemotherapy with the possibility of allogeneic stem cell transplantation. The encoded fusion protein may be important for leukemogenesis; clinical studies show that dasatinib has an antileukemic effect in combination with steroids alone or intensive chemotherapy.Areas covered: Relevant publications were identified through literature searches (the used terms being acute lymphoblastic leukemia plus dasatinib) in the PubMed database. We searched for original articles and reviews describing the pharmacology and clinical use of dasatinib in ALL with BCR-ABL1. The mechanism of action, pharmacology and clinical study findings are examined.Expert opinion: Dasatinib is associated with a high complete remission rate in ALL when used alone and in combination with steroids or intensive chemotherapy. However, mutations at T315 and F317 are associated with dasatinib resistance. Overall toxicity has been acceptable in these studies and no unexpected toxicity was observed. It is not known whether the antileukemic effect of dasatinib differs between subsets of BCR-ABL1+ patients or is attributed to inhibition of the fusion protein alone, or a combined effect on several kinases, and whether dasatinib-containing combination treatment should be preferred in these patients instead of other emerging strategies, e.g. monoclonal antibodies.Trial registration: ClinicalTrials.gov identifier: NCT02744768.Trial registration: ClinicalTrials.gov identifier: NCT02143414.Trial registration: ClinicalTrials.gov identifier: NCT02494882.Trial registration: ClinicalTrials.gov identifier: NCT02420717.Trial registration: ClinicalTrials.gov identifier: NCT02815059.Trial registration: ClinicalTrials.gov identifier: NCT00103701.Trial registration: ClinicalTrials.gov identifier: NCT00337454.Trial registration: ClinicalTrials.gov identifier: NCT00816283.Trial registration: ClinicalTrials.gov identifier: NCT03564470.Trial registration: ClinicalTrials.gov identifier: NCT03414450.Trial registration: ClinicalTrials.gov identifier: NCT01256398.Trial registration: ClinicalTrials.gov identifier: NCT00792948.Trial registration: ClinicalTrials.gov identifier: NCT00036738.Trial registration: ClinicalTrials.gov identifier: NCT01883219.Trial registration: ClinicalTrials.gov identifier: NCT03624530.Trial registration: ClinicalTrials.gov identifier: NCT01643603.Trial registration: ClinicalTrials.gov identifier: NCT01643603.Trial registration: ClinicalTrials.gov identifier: NCT02888990.Trial registration: ClinicalTrials.gov identifier: NCT02888977.

Modelling ponatinib resistance in tyrosine kinase inhibitor-naïve and dasatinib resistant BCR-ABL1+ cell lines.

TKI resistance remains a major impediment to successful treatment of CML. In this study, we investigated the emerging modes of ponatinib resistance in TKI-naïve and dasatinib resistant BCR-ABL1+ cell lines. To investigate potential resistance mechanisms, ponatinib resistance was generated in BCR-ABL1+ cell-lines by long-term exposure to increasing concentrations of ponatinib. Two cell lines with prior dasatinib resistance demonstrated BCR-ABL1 kinase domain (KD) mutation(s) upon exposure to ponatinib. In one of these cell lines the T315I mutation had emerged during dasatinib exposure. When further cultured with ponatinib, the T315I mutation level and BCR-ABL1 mRNA expression level were increased. In the other cell line, compound mutations G250E/E255K developed with ponatinib exposure. In contrast, the ponatinib resistant cell lines that had no prior exposure to other TKIs (TKI-naïve) did not develop BCR-ABL1 KD mutations. Rather, both of these cell lines demonstrated Bcr-Abl-independent resistance via Axl overexpression. Axl, a receptor tyrosine kinase, has previously been associated with imatinib and nilotinib resistance. Ponatinib sensitivity was restored following Axl inhibition or shRNA-mediated-knockdown of Axl, suggesting that Axl was the primary driver of resistance and a potential target for therapy in this setting.

Abstract A04: Dasatinib shows different cytotoxicity in triple-negative breast cancer cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is defined by the lack of significant expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Thus, TNBC is resistant to existing molecular targeted drugs such as trastuzumab or tamoxifen, and has a worse prognosis than non-TNBC. In addition, recent studies suggest that TNBC may represent a heterogeneous group of tumors with regards to gene expression, prognosis, and chemotherapy response. Although various molecular targeted drugs for TNBC have been attempted in clinical trials, almost all were negative outcomes because TNBC patients have a heterogeneous behavior. Therefore, in order to establish an efficient therapy, it is important to investigate the difference in response to molecular targeted drugs. From this viewpoint, we focused on dasatinib (Bcl-Abl/Src kinase inhibitor, approved in chronic myeloid leukemia) for the reason that it has been shown to be effective in some patients, even though it failed in clinical trials of unselected TNBC patients in the past. Here, to elucidate the mechanism of different response in dasatinib treatment on TNBC, we attempted to analyze the effects of dasatinib on cytotoxicity in some kinds of TNBC cell lines and to analyze the expression of targeted protein (Src) expression as a first step for elucidating the molecular mechanism of some kinds of TNBC cell lines. Methods: In this study, we used four TNBC cell lines such as HCC1806, HCC1395, MDA-MB-453, and MDA-MB-436, and one non-TNBC cell line, MCF-7. These cells were treated with a range of concentrations of dasatinib (0.001, 0.01, 0.1, 1, 10, or 100 μM) for 72 h. After treatment, cytotoxicity was evaluated by WST-8 assay and molecular expression was examined by Western blotting. Results and Discussion: WST-8 assay revealed that dasatinib showed high cytotoxicity in MCF7, HCC1395, and HCC1806 (IC50 were 0.162, 0.490, and 0.110 μM, respectively). Compared to these three cell lines, dasatinib showed low cytotoxicity in MDA-MB-453 and MDA-MB-436 (IC50 were 6.93 and 7.59 μM, respectively). Then, to investigate the mechanism that the dasatinib showed the different cytotoxicity among these cell lines, the expression of Src, target of dasatinib was examined. The higher expression of Src was observed in three cell lines, indicating relatively high cytotoxicity for dasatinib than the others. Although expression of Src was not observed in MDA-MB-453, that in MDA-MB-436 was the same extent as MCF7 despite low cytotoxicity for dasatinib. Thus, we evaluated the protein activity downstream of the Src pathway. Regarding the three cell lines in which dasatinib showed relatively high cytotoxicity, the phosphorylation of Akt after dasatinib treatment was degraded. However, there was no significant change of Akt activity in the two cell lines that showed relatively low cytotoxicity on dasatinib treatment. Now, in order to elucidate the molecular mechanism of dasatinib resistance observed in MDA-MB-436, we focus on autophagy that has reported to be suppressed by Src. In this regard, we indicated that the expression of LC3-II, known as a marker for autophagy, was higher in MDA-MB-436 than that of other cells in this study. Therefore, we are trying to clarify the relationship between Src and LC3-II. Conclusions: Each TNBC cell line shows the different response to dasatinib and the different Src expression, suggesting that the efficacy of dasatinib could not be dependent on the expression of Src. Thus, to clarify the mechanism of differences of cytotoxicity caused from a variety of TNBC cell lines, it is necessary to analyze more protein expressions upstream or downstream of the Src pathway. Citation Format: Yuya Haga, Kazuma Higashisaka, Lili Yang, Naoki Sekine, Kazuya Nagano, Yasuo Tsutsumi. Dasatinib shows different cytotoxicity in triple-negative breast cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A04.