Dasatinib-treated Mice Research Articles

The tyrosine kinase inhibitor Dasatinib reduces cardiac steatosis and fibrosis in obese, type 2 diabetic mice

BackgroundCardiac steatosis is an early yet overlooked feature of diabetic cardiomyopathy. There is no available therapy to treat this condition. Tyrosine kinase inhibitors (TKIs) are used as first or second-line therapy in different types of cancer. In cancer patients with diabetes mellitus, TKIs reportedly improved glycemic control, allowing insulin discontinuation. They also reduced liver steatosis in a murine model of non-alcoholic fatty liver disease. The present study aimed to determine the therapeutic effect of the second-generation TKI Dasatinib on lipid accumulation and cardiac function in obese, type 2 diabetic mice. We also assessed if the drug impacts extra-cardiac fat tissue depots.MethodsTwo studies on 21-week-old male obese leptin receptor mutant BKS.Cg-+Leprdb/+Leprdb/OlaHsd (db/db) mice compared the effect of Dasatinib (5 mg/kg) and vehicle (10% DMSO + 90% PEG-300) given via gavage once every three days for a week or once every week for four weeks. Functional and volumetric indices were studied using echocardiography. Post-mortem analyses included the assessment of fat deposits and fibrosis using histology, and senescence using immunohistochemistry and flow cytometry. The anti-adipogenic action of Dasatinib was investigated on human bone marrow (BM)-derived mesenchymal stem cells (MSCs). Unpaired parametric or non-parametric tests were used to compare two and multiple groups as appropriate.ResultsDasatinib reduced steatosis and fibrosis in the heart of diabetic mice. The drug also reduced BM adiposity but did not affect other fat depots. These structural changes were associated with improved diastolic indexes, specifically the E/A ratio and non-flow time. Moreover, Dasatinib-treated mice had lower levels of p16 in the heart compared with vehicle-treated controls, suggesting an inhibitory impact of the drug on the senescence signalling pathway. In vitro, Dasatinib inhibited human BM-MSC viability and adipogenesis commitment.ConclusionsOur findings suggest that Dasatinib opposes heart and BM adiposity and cardiac fibrosis. In the heart, this was associated with favourable functional consequences, namely improvement in an index of diastolic function. Repurposing TKI for cardiac benefit could address the unmet need of diabetic cardiac steatosis.

Dasatinib Ointment Promotes Healing of Murine Excisional Skin Wound.

Dasatinib, a tyrosine kinase inhibitor, has been shown to produce anti-inflammatory activity and impair vascular integrity in vivo, including during skin wound healing, potentially promoting the repair process. Given that dasatinib is a lipophilic small molecule capable of penetrating skin, topical dasatinib might provide benefits in wound healing. In the present study, we investigated the impact of dasatinib ointments in skin wound healing in mice. A full thickness excisional skin wound (4 mm diameter) was generated on the shaved dorsum of eight-week-old C57BL/6 mice. Dasatinib ointment (0.1 or 0.2% w/w) or ointment base was applied twice daily (every 12 h) for 10 days. Elizabethan collars were used to prevent animal licking. The wound size was monitored daily for 14 days. The results showed that dasatinib ointments, particularly 0.1% dasatinib, promoted a 16-23% reduction in wound size (p < 0.05) during day 2 to day 6 postinjury compared to controls. Immunohistochemistry analyses demonstrated a reduction in wound neutrophils (38% reduction, p = 0.04), macrophages (47% reduction, p = 0.005), and tumor necrosis factor-α levels (73% reduction, p < 0.01), together with an induction of vascular leakage-mediated fibrin(ogen) accumulation (2.5-fold increase, p < 0.01) in the wound during day 3 postinjury (an early phase of repair) in 0.1% dasatinib-treated mice relative to control mice. The anti-inflammatory and vascular hyperpermeability activities of dasatinib were associated with an enhanced healing process, including increased keratinocyte proliferation (1.8-fold increase in Ki67+ cells, p < 0.05) and augmented angiogenesis (1.7-fold increase in CD31+ area, p < 0.05), compared to the ointment base-treated group. Following treatment with 0.2% dasatinib ointment, minor wound bleeding and scab reformation were observed during the late phase, which contributed to delayed healing. In conclusion, our data suggest that dasatinib ointment, mainly at 0.1%, promotes the repair process by reducing inflammation and producing a local and temporal vascular leakage, leading to an increase in fibrin(ogen) deposition, re-epithelialization, and angiogenesis. Therefore, topical dasatinib might be a potential novel candidate to facilitate skin wound healing.

CML-074 Pathogenesis of Tyrosine Kinase Inhibitor–Associated Vascular Toxicity in Chronic Myeloid Leukemia

Evidence has emerged demonstrating an association between treatment with certain tyrosine kinase inhibitors (TKIs) and vascular adverse events (VAEs) in chronic myeloid leukemia (CML). An increase in VAEs has been documented with ponatinib, nilotinib, and dasatinib but not with imatinib and bosutinib. We used an endothelial cell (HUVEC) model and an in-vivo hindlimb ischemia mouse model to study TKI-associated VAEs. We show that compared with control-treated cells, ponatinib increased HUVEC apoptosis by 97% and inhibited their VEGF-induced migration by 35%. HUVEC tube formation was significantly abrogated by ponatinib as evidenced by an 87% reduction in tube number, 68% reduction in segment length, 50% reduction in junction number, and 55% increase in the occurrence of isolated segments. Ponatinib's suppressive effect on tube formation was partially rescued by VEGFR2 overexpression, a known ponatinib off-target. Dasatinib also dramatically disrupted tube formation as evidenced by a 97% reduction in tube number, 95% reduction in segment length, 88% reduction in junction number, and 300% increase in the occurrence of isolated segments. Nilotinib was associated with reduced cell viability, whereas imatinib and bosutinib did not affect these processes. Furthermore, HUVECs treated with ponatinib, dasatinib, and nilotinib revealed a differential angiogenesis-related gene expression pattern. The expression profile of imatinib- and bosutinib-treated cells was similar to that of control-treated cells. In our in-vivo model, unilateral femoral artery excision was performed to induce ischemia and stimulate new blood vessel formation in TKI-treated mice. Imatinib-treated mice showed a similar recovery pattern to that of control mice as assessed via Doppler imager. Bosutinib- and dasatinib-treated mice showed an initial delayed recovery but reached an overall recovery of 85% within 28 days. In contrast, ponatinib-treated mice reached overall perfusion of 60% around day 7 without further improvement. These results are in line with the clinical VAE profiles of ponatinib, dasatinib, and nilotinib, implicating endothelial toxicity related to these TKIs and supporting a direct effect of these TKIs on vascular cells. However, the in-vivo effect of these TKIs on endothelial pathogenesis requires further investigation. Understanding TKI-induced VAE pathogenesis could contribute to the future development of safer next-generation TKIs and could potentially influence TKI-personalized tailoring for CML patients.

Abstract 5341: Mechanisms driving resistance in Group 3 and recurrent/disseminated medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for 20% of childhood brain tumors. Among the 4 molecular MB subtypes, Group 3 and 4 MBs have the worst prognosis. The biology of recurrence and dissemination in MB is less understood. Overexpression of platelet-derived growth factor receptor (PDGFR) has been associated with metastatic MB, and PDGFR activity promotes MB migration. PDGFR-induced migration is regulated by Src kinase. Src kinase activity is high in MB and Src kinase inhibition restricts tumor growth. Group 3 MB demonstrates high PDGFRA expression. Aim: Our objective was to evaluate if molecular inhibition of PDGFR or Src kinase will control metastatic progression/dissemination in Group 3 MB. Methods: We treated a panel of 4 patient-derived MB cell lines with dasatinib, which can inhibit BCRABL, c-KIT, PDGFR and Src kinase. Dasatinib dose required to inhibit c-KIT and PDGFR is higher (50-100 nM) than that needed for Src inhibition (1-10 nM). Results: Proliferation assay with dasatinib (1-10000nM) performed on all 4 cell lines did not demonstrate any inhibition of proliferation except at 10000nM dose. We treated all 4 MB cell lines with higher dose range of dasatinib (625-10000nM). One cell line did not respond to dasatinib, remaining cell lines demonstrated proliferation inhibition over 13 days between 625-5000nM. There were 2 interesting observations. Firstly, cell line which required lowest dose (625nM) was a recurrent MB, while paradoxically, its paired parental cell line (non-recurrent/treatment-naïve) responded to proliferation inhibition only at higher 5000nM dose. IC50 of parental cell line was 38-times higher than recurrent cell line. We postulated that recurrent MB may be responsive to dasatinib. However, dasatinib-treated recurrent cells demonstrated minimal changes in cell cycle phases (IC50 day 4 dose 187.4nM) and apoptotic cell death (187.4nM and 293.2nM) compared to DMSO-treated control. Since Group 3 MB demonstrates high PDGFRA expression, we employed a patient-derived orthotopic xenograft (PDOX) model of Group 3 MB to investigate the in-vivo response to dasatinib. The 2nd interesting observation was that dasatinib-treated mice exhibited earlier neuro-symptoms, more rapid tumor growth and clinical deterioration compared to control mice. However, overall animal survival time was not statistically significant between treated versus untreated groups (p=0.24 Log-rank test). Conclusions: Recurrent cells were more sensitive to dasatinib in-vitro compared to paired parental cells. In-vivo, mice treated with dasatinib developed neuro-symptoms faster than controls. It is currently not known if recurrent MB cells demonstrating early dasatinib-sensitivity to proliferation inhibition, escaping cell death, can formulate an in-vivo mechanism to drive more rapid tumor progression/dissemination as observed in our Group 3 MB Model. Citation Format: Shiying Huang, Jie-Ling Pan, Qi Lin, YuChen Du, Jack Su, Angela Major, M. Tarek Elghetany, Kam-Man Hui, Xiaonan Li, Wan-Yee Teo. Mechanisms driving resistance in Group 3 and recurrent/disseminated medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5341.

Dasatinib protects against acute respiratory distress syndrome via Nrf2-regulated M2 macrophages polarization.

Dasatinib, a tyrosine kinase inhibitor, has a protective effect on experimental acute respiratory distress syndrome (ARDS). This study investigated the effect and mechanism of dasatinib in ARDS. C57BL/6 mice were administered with dasatinib (1 and 10mg/kg) after lipopolysaccharide (LPS) treatment to evaluate the effect of dasatinib on white blood cells (WBC), neutrophils, lymphocytes and macrophages in bronchoalveolar lavage fluid (BALF). The levels and mRNA expressions of inflammation-related cytokines in lung tissues and RAW 264.7 cells were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. The protein expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1) were determined by Western blot. MTT assay was performed to detect the viability of RAW 264.7 cell. Rescue experiments were used to assess the effect of Nrf2 silencing on the LPS- and dasatinib-treated mice. Under LPS treatment, levels of the WBC, neutrophils, lymphocytes and macrophages in BALF and mRNA expressions of IL-6, TNF-α and IL-10 as well as expression of iNOS were increased, but the expression of arginase-1 was inhibited, while no obvious changes of the protein expressions of Nrf2 and HO1 were observed. Dasatinib partially reversed the effects of LPS above, and further promoted the mRNA expression of IL-10 and the protein expressions of Nrf2 and HO1, while Nrf2 silencing counteracted the effect of dasatinib. Dasatinib induced the polarization of M2 subtype of macrophages and alleviated LPS-induced ARDS through activating Nrf2 signaling pathway, which may provide a new strategy for the treatment of ARDS.

Dasatinib protects humanized mice from acute HIV-1 infection

HIV-1 infection remains incurable despite the efficient combined antiretroviral therapy (cART) due to the formation of long-lived viral reservoirs that are mostly settled in CD4+T cells and maintained by homeostatic proliferation. The use of cytostatic drugs such as tyrosine kinase inhibitors (TKIs) as adjuvants to cART could be helpful to avoid the reservoir establishment and replenishment. We determined previously that TKI dasatinib, which is successfully used for treating chronic myeloid leukemia (CML), shows antiviral effect against HIV-1 infection of CD4+ T cells in vitro. HIV-infected subjects that developed CML may safely combine long-term treatment with TKIs and cART but there is no information about the effect of dasatinib on HIV-1 reservoir in vivo. Therefore, we analyzed the ability of dasatinib to protect NSG mice engrafted with human CD34+ hematopoietic stem cells from HIV-1 infection. Mice were randomly assigned to two groups that received dasatinib or placebo daily by oral gavage. After five days, all mice were infected intraperitoneally with HIV-1 and followed up for 21 days in the absence of cART. Daily administration of dasatinib decreased viral and proviral load in all treated mice, showing in 40% of these mice undetectable viral RNA or DNA in blood. Proviral HIV-1 DNA in gut-associated lymphoid tissue (GALT) was also reduced in all dasatinib-treated mice and under the limit of detection in one of these mice. Finally, treatment with dasatinib modified the distribution of CD4+ and CD8+ T-cell subpopulations, delaying their differentiation into memory T-cell subsets that are a major component of the viral reservoir. In conclusion, dasatinib afforded protection of NSG mice from HIV-1 intraperitoneal infection in the absence of cART.

The Yin and Yang of Tyrosine Kinase Inhibition During Experimental Polymicrobial Sepsis.

Neutrophils are the first cells of our immune system to arrive at the site of inflammation. They release cytokines, e.g., chemokines, to attract further immune cells, but also actively start to phagocytose and kill pathogens. In the case of sepsis, this tightly regulated host defense mechanism can become uncontrolled and hyperactive resulting in severe organ damage. Currently, no effective therapy is available to fight sepsis; therefore, novel treatment targets that could prevent excessive inflammatory responses are warranted. Src Family tyrosine Kinases (SFK), a group of tyrosine kinases, have been shown to play a major role in regulating immune cell recruitment and host defense. Leukocytes with SFK depletion display severe spreading and migration defects along with reduced cytokine production. Thus, we investigated the effects of dasatinib, a tyrosine kinase inhibitor, with a strong inhibitory capacity on SFKs during sterile inflammation and polymicrobial sepsis in mice. We found that dasatinib-treated mice displayed diminished leukocyte adhesion and extravasation in tumor necrosis factor-α-stimulated cremaster muscle venules in vivo. In polymicrobial sepsis, sepsis severity, organ damage, and clinical outcome improved in a dose-dependent fashion pointing toward an optimal therapeutic window for dasatinib dosage during polymicrobial sepsis. Dasatinib treatment may, therefore, provide a balanced immune response by preventing an overshooting inflammatory reaction on the one side and bacterial overgrowth on the other side.

Dasatinib Changes Immune Cell Profiles Concomitant with Reduced Tumor Growth in Several Murine Solid Tumor Models

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157-69. ©2017 AACR.

Abstract PR02: Investigating the use of tyrosine kinase inhibitors in Ph-like ALL.

Abstract Ph-like or BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (B-ALL) is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of the lymphoid transcription factor IKZF1, and poor outcome. The prevalence of Ph-like ALL rises with age from 10% in standard risk childhood ALL to over 25% in young adults with ALL. We recently performed genomic profiling of over 1700 B-ALL cases and next-generation sequencing of over 150 Ph-like ALL cases and identified alterations targeting 18 kinase or cytokine receptor genes, including rearrangement, sequence mutation and copy number alterations. The aim of this study was to determine the role of these kinase alterations in the development of Ph-like B-ALL, to define the signaling pathways that are activated, and to assess sensitivity to tyrosine kinase inhibitors (TKIs). To investigate the functional role of kinase fusions in Ph-like ALL, we introduced RCSD1-ABL2, SSBP2-CSF1R or PAX5-JAK2 into interleukin-7 dependent Arf-/- mouse pre-B cells expressing the dominant negative isoform of IKAROS, IK6. ABL2 (Abelson-related gene, ARG), a homolog of ABL1, has been implicated in the development of solid tumors, but rarely identified as a rearrangement partner in ALL. CSF1R (encoding the macrophage colony stimulating receptor) regulates the survival and differentiation of macrophages, and is not normally expressed in lymphoid cells. Each fusion tested conferred cytokine independent growth in vitro. Furthermore, recipient mice transplanted with pre-B cells expressing RCSD1-ABL2 or SSBP2-CSF1R developed ALL with a pre-B immunophenotype (CD43+, B220+, CD19+, BP-1+ and IgM-) and a median latency of 36 and 40 days respectively, implicating these kinases for the first time in the development of B-ALL. To assess activation of kinase signaling pathways, we performed phosphoflow cytometry analysis of Arf-/- pre-B cells or human leukemic cells harvested from xenografted mice expressing ETV6-ABL1, RANBP2-ABL1, PAG1-ABL2, RCSD1-ABL2, SSBP2-CSF1R, IGH-EPOR, ATF7IP-JAK2 and PAX5-JAK2. In both models we observed distinct patterns of signaling pathway activation and TKI sensitivity between the different fusions. Cells expressing ABL1-class kinase fusions (ABL1, ABL2, CSF1R and PDGFRB) showed activation of pSTAT5 that was inhibited with imatinib or dasatinib. Phosphorylation of CRKL, a known target of ABL1 and ABL2, was only observed in cells expressing these fusions. In contrast, leukemic cells harboring ATF7IP-JAK2, PAX5-JAK2 or IGH-EPOR showed activation of pSTAT5 that was only attenuated with the JAK2 inhibitor, ruxolitinib. This TKI response profile was confirmed by cytotoxicity assays, with ABL-class fusions being sensitive to dasatinib (IC50 range 1-2nM), whilst cases with ATF7IP-JAK2 and EPOR rearrangement responded to ruxolitinib, with IC50 values of 500nM and 850nM respectively. To further examine the utility of TKI therapy in Ph-like B-ALL, we investigated the in vivo efficacy of dasatinib in four xenograft models of ETV6-ABL1, RCSD1-ABL2, PAG1-ABL2 or SSBP2-CSF1R ALL. Dasatinib (20mg/kg/day p.o) or vehicle were commenced at measurable engraftment in peripheral blood (human CD45+/CD19+ cells &amp;gt;5%) and treatment ceased when leukemic burden exceeded 70% in vehicle treated mice. In all cases, dasatinib significantly reduced the leukemic burden and splenic weight compared to vehicle treated mice. Furthermore, phosphorylation of STAT5 was attenuated in cells harvested from dasatinib-treated mice compared to vehicle, indicating on target effect of the drug. Together, these data provide the first mouse models of Ph-like B-ALL, and show that despite the diversity of alterations identified, they activate distinct signaling pathways that can be targeted with a limited panel of FDA-approved agents. Prospective clinical trials that identify and direct Ph-like ALL patients to the appropriate TKI therapy may improve the treatment outcome for this high-risk subtype. Citation Format: Kathryn G. Roberts, Yung-Li Yang, Debbie Payne-Turner, Richard C. Harvey, I-Ming Chen, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl L. Willman, Stephen P. Hunger, Charles G. Mullighan. Investigating the use of tyrosine kinase inhibitors in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR02.

Abstract 159: CD90(+) intraperitoneal mesothelial-like cells (MLC) promote peritoneal metastasis by forming a tumor permissive microenvironment

Abstract Background: Peritoneal cavity is a common metastatic site of gastrointestinal as well as ovarian cancer, but the mechanisms to develop peritoneal metastasis has not been fully elucidated. Method: Peritoneal fluids were obtained in patients with liver cirrhosis or gastrointestinal cancer, and cells were cultured on collagen-coated plate and their characteristics were functionally analyzed both in vitro and in vivo. Results: We found that CD90(+)CD45(-) cells exist as a minor population (0.01∼6.81%) in human peritoneal fluids. However, those cells vigorously grew in culture on type I collagen-coated plate, showing the morphology of mesothelial cells, and names as mesothelial-like cells (MLC). The cultured MLC, when injected into the abdominal cavity of nude mice whose peritoneum was scratched by mechanical scraping, were incorporated into the damaged peritoneum. Interestingly, the MLC possessed the potential to differentiate into adipocytes, osteocytes, and chondrocytes in appropriate culture conditions. Moreover, the MLC markedly suppressed T cell proliferation stimulated with coated anti-CD3 mAb. Those findings are identical to the characteristics reported for mesenchymal stem cells (MSC). Moreover, by the stimulation of TGF-β, the MLC highly expressed type I collagen, vimentin, α-smooth muscle actin (SMA) and Fibroblast activated protein (FAP)-α, which are characteristics of activated myofibroblasts. From these facts, MLC with CD45(-)CD90(+) phenotype are supposed to be a intraperitoneal MSC. Then, we examined the possible role of the MLC on the development of peritoneal metastasis. Intraperitoneal co-injection of PKH26 stained MLC with human gastric cancer cell line, MKN45, significantly enhanced the rate of metastatic formation on peritoneum in nude mice. Histological examination revealed that many MLC are engrafted in metastatic nodules and they were mainly located at fibrous area in peritoneal nodules. Immunostaining showed that the MLC in peritoneal nodules are positive for type I collagen. Finally, Dasatinib, a potent tyrosine kinase inhibitor, strongly inhibited the in vitro proliferation of MLC, but had no effects on MKN45. However, oral administration of Dasatinib significantly inhibited the development of peritoneal metastasis of MKN45, and the tumor nodules developed in Dasatinib-treated mice showed significantly less fibrous components. Conclusion: These facts strongly suggests that intraperitoneal free floating MLC play a positive role on the development of peritoneal metastasis, possibly though the production of tumor permissive microenvironment and thus the functional blockade of MLC can be a reasonable strategy to treat peritoneal recurrences in abdominal malignancy. Citation Format: Joji Kitayama, Shiegenobu Emoto, Hironori Yamaguchi, Hironori Ishigami, Toshiaki Watanabe. CD90(+) intraperitoneal mesothelial-like cells (MLC) promote peritoneal metastasis by forming a tumor permissive microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2014-159

Combined Effects of a Pan-ABL1 Kinase Inhibitor, Ponatinib and Dasatinib Against T315I Mutant Forms of BCR-ABL1: In Vitro and In Vivo Studies

Abstract 2482Ponatinib is a third-generation ABL1 kinase inhibitor optimized using structure-based drug design to bind the inactive, DFG-out conformation of wild-type (WT)-BCR-ABL1 and T315I BCR-ABL1. Ponatinib provides an effective in minimizing resistance, however, certain BCR-ABL1 compound mutations such as Y235H/T315I and E255K/T315I are predicted to be recalcitrant to ponatinib. Combining ponatinib and dasatinib may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant compound mutations. In the present study, we investigated the combined effects of ponatinib and dasatinib in mutant forms of BCR-ABL1-expressing cells. Co-treatment with ponatinib and dasatinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to ponatinib and dasatinib including BaF3 cells expressing T315I. Initially, we performed random mutagenesis for BCR-ABL1 experiments (Blood, 109; 5011, 2007). Single-agent ponatinib completely suppressed outgrowth on resistant clones at 40 nM. As the immediate clinical application of ponatinib is in refractory BCR-ABL1 positive leukemia patients harboring T315I BCR-ABL1 mutation, we next performed resistant screens starting from T315I BCR-ABL1 BaF3 cells to identify BCR-ABL1 compound mutations. Compared with single agents, the combination of ponatinib and dasatinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of ponatinib (2 micro M), the compound mutation narrowed to Y253H/E225K/T315I in single cell by direct sequencing. To assess the mechanism of combination effect between ponatinib and dasatinib on T315I BCR-AB1L compound mutation-expressing cells, we used RNA interference to determine whether reduction of dasatinib off-target molecules such as KHS, Raf-1, B-Raf, ACK-1, FAK, EPHB4 may affect the growth inhibition. BaF3 cells expressing T315I BCR-ABL1 pretreated with Raf-1or EPHB4 siRNA showed enhanced growth inhibition with ponatinib. These results demonstrate that the enhanced growth inhibition by ponatinib and dasatinib in T315I-expressing cells may be mediated by Raf-1 and EPHB4. To assess the in vivo efficacy of ponatinib and dasatinib, athymic nude mice were injected s.c. with BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 5 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, ponatinib (30 mg/kg; q.d.), dasatinib (10 mg/kg; q.d.), ponatinib (30 mg/kg; q.d.) + dasatinib (10 mg/kg; q.d.). The ponatinib and dasatinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or ponatinib- or dasatinib-treated mice. Histopathologic analysis of tumor tissue from ponatinib+dasatinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. This study provides comparative drug combination profiling analysis of the effect of ponatinib and dasatinib, these two compounds are clinical active for BCR-ABL1-positive leukemia. Both agents commonly target a number of important molecular pathways that regulate cell growth and survival. A single proapoptotic or antiproliferative pathway may not be critical for the therapeutic effects of ABL1 kinase inhibitors. The present findings have important implications for the clinical use of ponatinib and dasatinib as anti-leukemia agent either alone or in combination with other agents. Disclosures:No relevant conflicts of interest to declare.

Dasatinib enhances megakaryocyte differentiation but inhibits platelet formation

AbstractDasatinib is a novel, potent, ATP-competitive inhibitor of Bcr-Abl, cKIT, and Src family kinases that exhibits efficacy in patients with imatinib-resistant chronic myelogenous leukemia. Dasatinib treatment is associated with mild thrombocytopenia and an increased risk of bleeding, but its biological effect on megakaryocytopoiesis and platelet production is unknown. In this study, we show that dasatinib causes mild thrombocytopenia in mice without altering platelet half-life, suggesting that it inhibits platelet formation. Conversely, the number of megakaryocytes (MKs) in the bone marrow of dasatinib-treated mice was increased and the ploidy of MKs derived from bone marrow progenitor cells in vitro was elevated in the presence of dasatinib. Furthermore, a significant delay in platelet recovery after immune-induced thrombocytopenia was observed in dasatinib-treated mice even though the number of MKs in the bone marrow was increased relative to controls at all time points. Interestingly, the migration of MKs toward a gradient of stromal cell–derived factor 1α (SDF1α) and the formation of proplatelets in vitro were abolished by dasatinib. We propose that dasatinib causes thrombocytopenia as a consequence of ineffective thrombopoiesis, promoting MK differentiation but also impairing MK migration and proplatelet formation.

Abstract A38: Caveolin-1 as a biomarker of drug response and therapeutic target in association with tyrosine kinase inhibitor treatment in prostate cancer models

Abstract We have shown that caveolin-1 (Cav-1) is overexpressed in metastatic prostate cancer (PCa) and that virulent PCa cells secrete biologically active Cav-1. Secreted Cav-1 can be taken up by adjacent PCa and tumor-associated endothelial cells and promote tumor angiogenesis. We investigated the effect of dasatinib and sunitinib on proliferation, tyrosine kinase (TK) phosphorylation, and downstream signaling pathways, including Cav-1 expression and secretion in hormone-refractory PCa cell lines (PC-3 and DU145) in vitro. Dasatinib inhibited proliferation of PC-3 and DU145 at doses from 0.05 to 5.0 μM; sunitinib inhibited their proliferation at doses from 0.2 to 20 μM. Dasatinib and sunitinib treatment yielded a dose-dependent reduction in phosphorylation of PDGFR (Y857), VEGFR2 (Y951), Akt (S473), and Cav-1 (Y14) in PC-3 and DU145 cells relative to that in controls. Further, dasatinib treatment of PC-3 and DU145 cells resulted in reduced phosphorylation of FAK (Y861) and Src (Y416). Sunitinib did not cause a similar effect on FAK in PC-3 and DU145 cells but did cause a reduction in Src phosphorylation of DU145, though not of PC-3 cells. It was interesting that dasatinib and sunitinib treatment of PC-3 and DU145 cells each inhibited the secretion of Cav-1 and caused moderate reduction in cellular Cav-1. To analyze the role of secreted Cav-1 as a biomarker of drug response and therapeutic target in the context of dasatinib and sunitinib treatment in vivo, we used PC-3 and DU145 subcutaneous xenograft models. PC-3 xenografts were treated with dasatinib (15 mg/kg q.d., p.o.), Cav-1 antibody (Ab; 10 μg, q.o.d., i.p.), or combined dasatinib and Cav-1 Ab, or vehicle alone. DU145 xenografts were treated with sunitinib (10 mg/kg q.d., p.o.), Cav-1 Ab (10 μg, q.o.d., i.p.), combined sunitinib and Cav-1 Ab, or vehicle alone. Treatment with either dasatinib or Cav-1 Ab produced significant tumor regression (P = 0.0072 and 0.0307, respectively) compared with that of vehicle or IgG. Combined dasatinib and Cav-1 Ab produced greater tumor regression than either treatment alone, but the differences did not achieve statistical significance. Similarly, treatment with either sunitinib or Cav-1 Ab induced significant DU145 tumor regression (P= 0.0004 and 0.0016, respectively) compared with that of vehicle or IgG. Combined sunitinib and Cav-1 Ab induced greater but not significantly different tumor regression than either treatment alone. We found it interesting that serum Cav-1 levels were significantly lower in dasatinib-treated mice than they were in vehicle-treated mice (P = 0.0271). Sunitinib treatment also led to lower serum Cav-1 levels than the vehicle did, but these differences were not statistically significant (P = 0.0871). An important note is that serum Cav-1 levels correlated positively with tumor growth (wet weight) in combined vehicle- and dasatinib-treated groups (r = 0.48, P = 0.031) and combined vehicle- and sunitinib-treated groups (r = 0.554, P = 0.0065). Overall, our results show that dasatinib and sunitinib can each inhibit PCa cell growth both in vitro and in vivo and that growth inhibition is associated with inhibition of TK phosphorylation and downstream signaling and suppression of Cav-1 secretion. We also found that reduced serum Cav-1 was associated with dasatinib and sunitinib treatment and that serum Cav-1 levels correlate positively with tumor growth. Moreover, we showed that systemic administration of Cav-1 Ab led to suppressed tumor growth in PCa xenografts. These results suggest roles for secreted/serum Cav-1 as a biomarker of drug response and as a therapeutic target in association with dasatinib or sunitinib treatment in PCa. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A38

Combined Effects of a Pan-Aurora Kinase Inhibitor MK-0457 and Dasatinib Against T315I Mutant Form of BCR-ABL: In Vitro and In Vivo Studies.

MK-0457 is a pan-Aurora kinase inhibitor that has firstly shown clinical activity in BCR-ABL positive leukemia patients harboring the T315I mutation. Combining MK-0457 with ABL kinase inhibitors may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant mutations. In the present study, we investigated the combined effects of MK-0457 and dasatinib in T315I mutant-expressing cells. Co-treatment with MK-0457and dasatinib caused significantly more inhibition of colony growth of primary leukemia cells and BaF3 cells expressing T315I BCR-ABL than either drug alone. In contrast, we did not observe the enhanced effects of MK-0457 and imatinib in T315I BCR-ABL-expressing cells. Treatment with 5 μM of MK-0457 for 48 hrs induced apoptosis in T315I BCR-ABL BaF3 cells, whereas, exposure to combination with 1.0 μM of MK-0457 and 50 nM of dasatinib exerted enhanced apoptotic effect. Combined treatment with MK-0457 and dasatinib also associated with more PARP cleavage, which is due to increased activation of caspase-3 and caspase-9 during apoptosis. Following co-treatment with MK-0457 and dasatinib caused more attenuation of the level of phospho-Stat5 and the downstream signal transducer, including Bcl-XL, Mcl-1, and cyclin D1. We also observed that combined treatment with MK-0457 and dasatinib enhanced the activation of p38MAP kinase and MAPKAP Kinase-2 (MK2). Pretreatment with p38MAP kinase or MP2 siRNA in T315I BCR-ABL BaF3 cells reduced the induction of apoptosis after MK-0457 and dasatinib exposure. These results suggest that p38MAP kinase and MK2 have a critical role for the induction of apoptosis after MK-0457 and dasatinib exposure. To assess the mechanism of combination effect between MK-0457 and dasatinib on T315I BCR-ABL-expressing cells, we used RNA interference to determine whether reduction of SRC-family kinases affected the growth inhibition. BaF3 cells expressing T315I BCR-ABL pretreated with Lyn or Hck siRNA showed enhanced growth inhibition with MK-0457. These results demonstrate that the enhanced growth inhibition by MK-0457 and dasatinib in T315I-expressing cells may be mediated by Lyn and Hck. To assess the in vivo efficacy of MK-0457 and dasatinib, athymic nude mice were injected i.v. with BaF3 cells expressing T315I mutant form of BCR-ABL. 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, MK-0457 (30mg/kg b.i.d.; ip for 5 days), dasatinib (10mg/kg q.d.; po for five days), MK-0457 (30mg/kg b.i.d.; ip for 5 days) + dasatinib (10mg/kg q.d.; po for five days). The treatment was repeated in every 3 weeks. The vehicle or dasatinib-treated mice died of a condition resembling acute leukemia by 28 days; the MK-0457 only-treated mice survived more than 56 days, and the combination of MK-0457 + dasatinib -treated mice survived more than 70 days. Histopathologic analysis of vehicle or dasatinib-treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from MK-0457 plus dasatinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, the present study shows that the combination of MK-0457 and dasatinib exhibits a desirable therapeutic index that can reduce the in vivo growth of T315I mutant form of BCR-ABL-expressing cells in an efficacious manner.