Family Tyrosine Kinase Inhibitor Research Articles

Gemcitabine and nab-paclitaxel combined with afatinib in metastatic pancreatic cancer – Results of a phase 1b clinical trial

PurposeThe combination of gemcitabine/nab-paclitaxel is an established standard treatment in the first-line treatment of metastatic ductal adenocarcinoma of the pancreas (mPDAC). Afatinib, an oral second-generation pan ErbB family tyrosine kinase inhibitor, has shown promising pre-clinical signs in the treatment of pancreatic cancer. The aim of this phase 1b trial was to determine the maximum tolerated dose (MTD) of afatinib in combination with gemcitabine/nab-paclitaxel in patients with mPDAC. MethodsTreatment naïve patients (≥18 years) with histologically proven mPDAC and good performance status (ECOG 0/1) were enrolled to receive gemcitabine/nab-paclitaxel in combination with afatinib. Treatment was continued until disease progression, or unacceptable toxicity. The primary endpoint MTD was determined using a 3 + 3 design. Treatment started at dose level 0 with intravenous gemcitabine/nab-paclitaxel 1000 mg/m2 / 125 mg/m2 (day 1, 8, 15 of a 28-day cycle) + oral afatinib 30 mg daily. At dose level + 1 afatinib was increased to 40 mg. Secondary endpoints included safety parameters and exploratory endpoints evaluated treatment efficacy. ResultsTwelve patients were included in this trial, and 11 patients were treated and analysed in the safety and full analysis set (FAS). At dose level 0 the first three patients did not experience a dose-limiting toxicity (DLT). At dose leve (DL) + 1 two patients experienced a DLT. Accordingly, enrolment continued at DL 0 with three more patients, of which one experienced DLT (skin rash ≥ CTCAE grade 3). Seven patients (63.6%) experienced at least one treatment-emergent serious adverse event (TESAE), with four patients (36.4%) experiencing TESAEs grade 3–5 related to the study medication. In the FAS, the objective response rate (ORR) was 36.4%, median progression-free survival (PFS) was 3.5 months and median overall survival in nine evaluable patients was 7.5 months. ConclusionsIn this phase 1b clinical trial, the MTD of gemcitabine/nab-paclitaxel (1000 mg/m2 / 125 mg/m2) and afatinib (30 mg) was established. In a cohort of 11 patients, the combination showed an acceptable safety profile.

Effect of Calcitriol in Inhibiting the Cancer Cell Growth and Promoting Apoptosis in ErbB2-positive Breast Cancer Cells.

Targeted therapies, specifically ErbB family tyrosine kinase inhibitors, have demonstrated potential for improving outcomes in patients with ErbB2-positive breast cancer. Despite their effectiveness, these therapies are associated with limitations, including high costs, side effects, drug resistance, lack of specificity, and toxicity. To overcome these challenges, drug repurposing has emerged as a promising strategy in breast cancer treatment. The aim of this investigation was to assess the influence of calcitriol on breast cancer cell lines expressing ErbB2 and comparing its effects with the conventional treatment, neratinib. We employed an MTT test to determine cell viability and utilized staining techniques to assess cell apoptosis. Flow cytometry was used to evaluate cell cycle arrest, while a scratch wound healing test was performed to examine cancer cell migration ability. Additionally, gene expression studies were conducted for calcitriol and neratinib to support our hypothesis regarding the ErbB2 gene. The repurposing of calcitriol demonstrated enhanced efficacy in suppressing cancer cell growth in ErbB2- positive breast cancer. Proportionally, calcitriol significantly reduced the viability of SK-BR-3 cells, similar to neratinib. Furthermore, calcitriol exhibited significant cytotoxicity against neratinib and substantially reduced breast cancer cell growth. These findings were corroborated by the wound healing assay, cell cycle arrest analysis, and gene expression studies, demonstrating comparable efficacy to the standard treatment, neratinib. The findings from this investigation offer compelling proof that highlights the promising role of calcitriol as an adjuvant drug with antiproliferative and antitumoral effects in the management of ErbB2-positive breast carcinoma patients. Therefore, we recommend further evaluation of calcitriol in clinical settings, particularly for the treatment of ErbB2-positive breast cancer, as it shows promise as a valuable therapeutic option.

Abstract 3099: Hnd-01: a potent inhibitor of wild type mutant BTK

Abstract Dasatinib is an oral dual BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Based on the in vitro and in vivo characterization studies of dasatanib, several metabolites were identified. Dasatinib and its metabolite 20 were the major circulating species identified which represented 25.5% and 12.5% of total radioactivity in human plasma respectively. Here, we report the identification of HND-01, a small molecule inhibitor of Bruton’s tyrosine kinase (BTK) which was achieved by modifying metabolite 20 of dasatinib. Bruton’s tyrosine kinase (BTK) is a non-receptor kinase and a key regulator of the B-cell receptor (BCR) signaling pathway. BCR is critical for proliferation and survival of leukemic cells in many B cell malignancies, including chronic lymphocytic leukemia (CLL). BTK is a key target for the development of small molecule inhibitors in B cell malignancies. There are several covalent BTK inhibitors including ibrutinib that have been approved. These covalent inhibitors bind with high affinity to Cys-481 in the active site of BTK. However, some patients develop acquired resistance to ibrutinib due to Cys-481 mutation in the BTK binding site. The acquired resistance to covalent inhibitors can be overcome with the design of new reversible BTK inhibitors. The reversible BTK inhibitors will bind to different sites other than the cysteine residue of BTK and inactivate BTK. These drugs would potentially be active in the presence or absence of the cysteine-to-serine mutation. HND-01, a novel and potent reversible BTK inhibitor inhibits wild type & C481S mutant BTK with IC50 values of 0.157 nM for WTBTK and 0.032 nM for C481SBTK. HND-01 inhibits K562 cell proliferation (IC50 0.183 nM) and it is more stable than dasatinib in human liver microsomes (in vitro clearance; 142 mL/min/kg for HND-01 vs 211 mL/min/kg for dasatinib). In an established human lymphoma efficacy model (TMD-8 model), HND-01 showed stronger tumor inhibition (80.6%) than Ibrutinib (58%). These data demonstrates that HND-01 is a BTK inhibitor that is more potent with potential desirable ADME properties than ibrutinib. HND-01 was filled for international patents, China patent application # 202080030953.6, US patent application #17/636,476, EU application # EP 20853834.8, and Canada application # CA 3148436. Citation Format: Jing Zhang, Ahmed A. Samatar, Yue Tan, Shiqing Pi, Zhigang Zhou, Yan Xu, Daihong Yang. Hnd-01: a potent inhibitor of wild type mutant BTK [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 3099.

Histamine Produced by Gram-Negative Bacteria Impairs Neutrophil’s Antimicrobial Response by Engaging the Histamine 2 Receptor

We found that histamine (10−9 M) did not have any effect on the in vitro capture of Escherichia coli by neutrophils but accelerated its intracellular killing. In contrast, histamine (10−6 M) delayed the capture of Escherichia coli by neutrophils and reduced the amounts of pHrodo zymosan particles inside acidic mature phagosomes. Histamine acted through the H4R and the H2R, which are coupled to the Src family tyrosine kinases or the cAMP/protein kinase A pathway, respectively. The protein kinase A inhibitor H-89 abrogated the delay in bacterial capture induced by histamine (10−6 M) and the Src family tyrosine kinase inhibitor PP2 blocked histamine (10−9 M) induced acceleration of bacterial intracellular killing and tyrosine phosphorylation of proteins. To investigate the role of histamine in pathogenicity, we designed an Acinetobacter baumannii strain deficient in histamine production (hdc::TOPO). Galleria mellonella larvae inoculated with the wild-type Acinetobacter baumannii ATCC 17978 strain (1.1 × 105 CFU) died rapidly (100% death within 40 h) but not when inoculated with the Acinetobacter baumannii hdc::TOPO mutant (10% mortality). The concentration of histamine rose in the larval haemolymph upon inoculation of the wild type but not the Acinetobacter baumannii hdc::TOPO mutant, such concentration of histamine blocks the ability of hemocytes from Galleria mellonella to capture Candida albicans in vitro. Thus, bacteria-producing histamine, by maintaining high levels of histamine, may impair neutrophil phagocytosis by hijacking the H2R.

Afatinib After Progression on Osimertinib in EGFR-Mutated Non-Small Cell Lung Cancer

After the development of acquired resistance to osimertinib, the standard-of-care treatment for advanced EGFR-mutated NSCLC is chemotherapy. Whether afatinib, a pan-ErbB family tyrosine kinase inhibitor, is active after progression on osimertinib is unknown. We conducted a single-institution retrospective analysis of patients with advanced EGFR-mutated NSCLC who received afatinib-containing therapy after progression on osimertinib. Kaplan-Meier analyses evaluated progression-free survival (PFS) and overall survival (OS) from initiation of afatinib. After progression on first (N=3) or second-line plus (N=12) osimertinib, 15 patients received afatinib monotherapy (N=3), afatinib and cetuximab (N=10), or afatinib and bevacizumab (N=2). The objective response rate was 6.7% and disease control rate was 53.3%. Median PFS was 2.5 months and median OS was 7.7 months. Median PFS of ≥ 6 months versus < 6 months on osimertinib was associated with a significantly greater median PFS on afatinib (4.0 versus 1.4 months; P=0.003), although there was no significant difference in median OS (9.3 versus 6.6 months; P=0.123). Best response of stable disease/partial response versus progressive disease on osimertinib was associated with a significantly greater median PFS on afatinib (3.4 versus 1.6 months; P=0.036) and a significantly greater median OS (8.7 versus 4.6 months; P=0.017). Afatinib-containing therapy had limited activity in patients with EGFR-mutated NSCLC after progression on osimertinib in this cohort of mostly second-line plus osimertinib. Response and longer PFS to prior osimertinib may be predictive of response to afatinib. Strategies based on osimertinib resistance mechanisms may further define the role of subsequent afatinib.

Expert consensus on the management of adverse events of ErbB family tyrosine kinase inhibitors in breast cancer

Human epidermal growth factor receptor-2 (HER-2) tyrosine kinase inhibitors (TKI) is the targeted drug of HER-2-positive breast cancer. Lapatinib, pyrotinib and neratinib, as ErbB family TKIs, have been approved by National Medical Products Administration and applied in the treatment of HER-2 positive breast cancer in China. The most common adverse effects (AEs) of TKI agents include diarrhea, drug-induced liver injury (DILI), nausea, vomiting, skin toxicity, cardiotoxicity and oral mucositis. The Breast Cancer Expert Group of Chinese Society of Clinical Oncology (CSCO) summarized the incidence and characteristics of AEs of TKI, evaluated the manifestations and severity of AEs, and formulated the consensus of the management of common AEs based on the clinical experiences and updated advances from domestic and abroad studies. This consensus aims to provide the practical strategy for clinicians in the management of ErbB-family TKI-related AEs in China, and eventually enhance the patients' compliance and improve the therapeutic efficacy.

Aberrant HER3 ligand heregulin-expressing head and neck squamous cell carcinoma is resistant to anti-EGFR antibody cetuximab, but not second-generation EGFR-TKI

The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is standard therapy for head and neck squamous cell carcinoma (HNSCC). However, most HNSCC tumors are resistant to it and require alternative treatments. Here, we explored the mechanism of cetuximab resistance and evaluated its clinical relevance in HNSCC. An unbiased comprehensive transcriptome analysis was performed on cetuximab-resistant HNSCC FaDuCR cells. The causative resistance genome was knocked down with siRNA, cell signaling was immunologically analyzed, and drug efficacy was evaluated in vitro and in vivo. The mRNA in situ hybridization (ISH) of the causative genome was performed using 28 excised HNSCC tumors and its relationship with cetuximab efficacy was analyzed. FaDuCR cells were resistant to cetuximab, whereas parental FaDu cells were susceptible to it. FaDuCR cells expressed consistently higher levels of phosphorylated Akt than FaDu cells despite cetuximab exposure. A comprehensive transcriptome analysis revealed that the HER3-ligand heregulin was upregulated in FaDuCR cells compared to FaDu cells. Heregulin knockdown in FaDuCR cells repressed HER3 and Akt phosphorylation and recovered cetuximab anticancer efficacy. In contrast, pan-HER family tyrosine kinase inhibitors such as afatinib decreased HER3 and Akt phosphorylation in FaDuCR cells and inhibited FaDuCR tumor growth. Two of the 28 HNSCC tumor samples presented aberrant heregulin expression comparable to that of FaDuCR cells and were resistant to cetuximab therapy. In HNSCC, heregulin-mediated HER3-Akt activation causes resistance to cetuximab but not to second-generation EGFR-tyrosine kinase inhibitors. Subpopulations with aberrant heregulin-expressing HNSCC might be resistant to cetuximab.

Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody-mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood-circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle formation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders.

Abstract 384: Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy

Abstract Cancer cells often survive by getting addicted to specific genes. In most cases, such genes function as an enzyme, especially kinases that are specifically activated in cancers by genetic aberrations; e.g., BCR-ABL fusion kinase found in chronic myelogenous leukemias and epidermal growth factor receptor (EGFR) with activating mutations found in non-small cell lung cancers (NSCLCs). These activated kinases would be an ideal target for cancer therapy and indeed imatinib and gefitinib displayed good clinical benefits to the patients in these diseases. However, only a limited population of cancer cells harbor such well-characterized driver genes which could be attacked by targeted drugs developed so far and there remain unmet medical needs to identify novel therapeutic targets and drugs for the rest of cancers. To this end, we exploited our original database of drug efficacy of &amp;gt;100 kinase inhibitors across a panel of 39 human cancer cell lines (JFCR39) to characterize each of the cell line in terms of susceptibility to these kinase inhibitors. Expectedly, cell lines harboring PIK3CA hotspot mutation and PTEN mutation were susceptible to PI3K and mTOR inhibitors, whereas those harboring KRAS or BRAF mutation to MEK inhibitors, respectively. Of note, we found four of the cells responding to a series of EGFR family tyrosine kinase inhibitors (TKIs) including gefitinib and lapatinib. Gefitinib exert selective effect on NSCLCs addicted to activated EGFR mutant; however, none of the four gefitinib-sensitive cell lines possessed such EGFR mutation. Of these, an NSCLC cell line NCI-H522 hardly expressed EGFR but overexpressed ERBB4 instead, while an ovarian cancer cell line SK-OV3 overexpressed ERBB2 as reported previously. Actually, treatment with a certain TKI in this class efficiently dephosphorylated ERBB4 in NCI-H522 along with inhibition of cell growth and suppression of downstream factor ribosomal S6 protein. Moreover, knockdown of ERBB family kinases revealed that cell growth was dependent on overexpressed ERBB4 in NCI-H522 cells. These results indicate that NCI-H522 cells exhibited susceptibility to EGFR family TKIs via inhibition of ERBB4. Of note, NCI-H522 cells exhibited hypersensitivity to three of these TKIs, sapitinib, PF-7775 and GW583340, as compared to gefitinib. Especially, GW583340 suppressed growth of NCI-H522 cells at the same or lower concentration than in HCC827 cells harboring an EGFR active mutation. Consistent with this observation, in vitro kinase assay revealed that sapitinib and PF-7775 exhibited ~10-fold higher ERBB4-inhibitory effect than gefitinib did while GW583340 displayed selective inhibition on ERBB4, with ~10-fold higher activity than that on activated EGFR. In conclusion, we identified NCI-H522 as an ERBB4-dependent cell line, and found ERBB4 as a novel therapeutic cancer target that could be attacked by ERBB4-directing TKIs. Citation Format: Noritaka Tanaka, Kanami Yamazaki, Yuko Uno, Yoshimi Ohashi, Yumiko Nishimura, Masaaki Sawa, Shingo Dan. Identification of a lung adenocarcinoma cell line addicted to ERBB4, an actionable target for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 384.

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BackgroundAstrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte‐expressed Gαi/o‐coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant‐induced production of glial cell line‐derived neurotrophic factor (GDNF) and matrix metalloproteinase‐9 (MMP‐9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP‐9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP‐9.Methods and ResultsTreatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration‐dependent manner. Amitriptyline‐induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline‐induced zymographic MMP‐9 activation in C6 cells. The amitriptyline‐induced zymographic MMP‐9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline‐induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP‐9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1.ConclusionsThe current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o/Src family tyrosine kinase, which leads to MMP‐9 activation and GDNF production.

Abstract 1622: In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models

Abstract Background: Recently, poly ADP ribose polymerase (PARP) inhibitors have shown potential to increase the sensitivity of platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies in BRCA or DNA repair pathway genes altered or WT condition (Basourakos et al., 2017; Gampenrieder et al., 2017). Src, a non-receptor tyrosine kinase, is involved in the regulation of cell proliferation, survival and apoptotic ability of cancer cells (Tryfonopoulos et al., 2011; Pusztai et al., 2014). Dasatinib (D) is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which has displayed anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011). Our current study examines whether combinational inhibition of Src pathway (D) would exhibit greater antitumor activity of PARP inhibitor [veliparib (V)] plus carboplatin (C) in TNBC xenograft models with different genetic back grounds. Methodology: a) We utilized MDA-MB-231, MDA-MB-468 and SUM149PT cells for in vivo study. b) TNBC cells were implanted subcutaneously into immune-compromised mice (NCr Nu/Nu, Taconic) to generate tumor xenografts. c) We evaluated the effects on tumor growth inhibition of D (12.5 mg/kg, i.p., Qd for 4 weeks) / V (25 mg/kg, oral, Bid for 5 days) / C (40mg/kg, i.v., once) as single agent or in combinations (Chan et al., 2012; De et al., 2014). d) For IHC studies, tumor tissue sections were stained with respective antibodies for proliferation, cell survival, angiogenesis and apoptosis, including: phospho-Src, Ki-67, phospho-S6 RP, phospho-Akt, phospho-ERK, CD31, cyclin D1 and cleaved caspase3. e) Student's t test was used for data analyses. Results: We observed that 1) MDA-MB-231(KRAS/BRAF mutated) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, the best tumor inhibition result was induced by triple combination (p &amp;lt; 0.004); 2) MDA-MB-468 (PTEN null) xenograft model: D alone or combined with V or C or in triple combination significantly inhibited tumor growth, the best tumor inhibition result was observed by triple combination (p &amp;lt; 0.00006); 3) SUM149PT (BRCA1 mutated, PTEN null) xenograft model: D alone or combined with V or C or in triple combination inhibited tumor growth, and triple combination, as well as D plus C, showed greater (p &amp;lt; 0.0025) than other combinations / single agents; 4) Single agent of D / V / C was well tolerated with less than 10% mean body weight loss compared with control groups in all models. Conclusion: Our in vivo study suggests that D may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC xenograft tumors with different genetic backgrounds. Immunohistochemical studies of xenograft tumor samples are ongoing, the results of which will be presented at the meeting. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer Carlson, Pradip De, Nandini Dey, Casey Williams, Brian Leyland-Jones. In vivo study of dasatinib in combination with veliparib plus carboplatin in triple-negative breast cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1622.

Abstract 1820: Overcoming drug resistance in NSCLC with SRC inhibitor

Abstract Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1820.

Dasatinib Reversibly Disrupts Endothelial Vascular Integrity by Increasing Non-Muscle Myosin II Contractility in a ROCK-Dependent Manner.

Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat chronic myeloid leukemia. Although very effective, patients taking dasatinib often display severe adverse effects, including pleural effusions and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to the underlying tissues might be involved.Experimental Design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance measurements, live-cell microscopy, wound healing, Western blot, and an in vivo model.Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted the disassembly of cell-cell contacts, altered cell-matrix contacts, and further altered the wound healing. A key observation is that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinib-induced reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin light chain and consequently activates the non-muscle myosin II.Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that probably contributes to adverse effects such as bleeding diathesis and pleural effusions. Clin Cancer Res; 23(21); 6697-707. ©2017 AACR.

Dasatinib related pericardial effusion requiring pericardial drainage

Introduction Dasatinib is an oral Bcr-Abl and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukaemia (ALL). Its common side effects include myelosuppression, oedema, diarrhea and nausea. It has also been associated with the formation of pleural and pericardial effusions. As a result, Dasatinib is to be avoided in patients with pre-existing effusions or predisposition to respiratory or cardiovascular disease. Case description A fit 62-year-old pilot with no relevant medical history was diagnosed with CML in 2014, and commenced on Dasatinib therapy (100mg OD). A subsequent trans-thoracic echocardiogram (TTE) revealed normal ventricles and cardiac valves. There was however a mild to moderate global pericardial effusion, without haemodynamic compromise. This was regularly monitored with TTEs and remained stable until May 2016, where it measured 2.1cm posteriorly around the LV and 1.0 cm around the RV. Due restrictions imposed by the Civil Aviation Authority in the UK, the patient was referred for pericardial window procedure, prior to being considered fit for flying. Conclusions Dasatinib is known to cause pleural and pericardial effusions. This has been reported in patients without any predisposing factors.(1) The link with pericardial effusions has been proven with robust statistical analysis.(2) No specific mechanism has been proposed but an immune mediated reaction or off target inhibition of growth factors may be involved.(3) Management includes dose interruption or reduction, and/or treatment with steroids.(3) Our case report re-enforces that Dasatinib is an important cause of pericardial effusion and TTE is the modality of choice for follow-up. Pericardial window and drainage may be needed in patients where this prohibits them from undertaking employment. Take-home message Dasatinib related pericardial effusions are a documented side effect of therapy. One should be vigilant in monitoring patients on the drug as effusions may progress over time and require intervention. TTE is the monitoring modality of choice. As far as we are aware this is the first case report for surgical intervention in a patient with Dasatinib induced pericardial effusion.

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases.

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2.

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.

Systematic drug sensitivity testing reveals synergistic growth inhibition by dasatinib or mTOR inhibitors with paclitaxel in ovarian granulosa cell tumor cells

ObjectiveResistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. MethodsThe responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. ResultsPatient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. ConclusionsWe used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high-throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options.

Src family tyrosine kinase inhibitors suppress Nav1.1 expression in cultured rat spiral ganglion neurons.

Src family kinases regulate neuronal voltage-gated Na(+) channels, which generate action potentials. The mechanisms of action, however, remain poorly understood. The aim of the present study was to further elucidate the effects of Src family kinases on Nav1.1 mRNA and protein expression in spiral ganglion neurons. Immunofluorescence staining techniques detected Nav1.1 expression in the spiral ganglion neurons. Additionally, quantitative PCR and western blot techniques were used to analyze Nav1.1 mRNA and protein expression, respectively, in spiral ganglion neurons following exposure to Src family kinase inhibitors PP2 (1 and 10 μM) and SU6656 (0.1 and 1 μM) for different lengths of time (6 and 24 h). In the spiral ganglion neurons, Nav1.1 protein expression was detected in the somas and axons. The Src family kinase inhibitors PP2 and SU6665 significantly decreased Nav1.1 mRNA and protein expression (p < 0.05), respectively, in the spiral ganglion neurons, and changes in expression were not dependent on time or dose (p > 0.05).

The pan-HER family tyrosine kinase inhibitor afatinib overcomes HER3 ligand heregulin-mediated resistance to EGFR inhibitors in non-small cell lung cancer

Afatinib is a second generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) characterized as an irreversible pan-human EGFR (HER) family inhibitor. Afatinib remains effective for a subpopulation of patients with non-small cell lung cancer (NSCLC) with acquired resistance to first generation EGFF-TKIs such as erlotinib. Heregulin activates HER3 in an autocrine fashion and causes erlotinib resistance in NSCLC. Here we examine whether afatinib is effective against heregulin-overexpressing NSCLCs harboring EGFR activating mutations. Afatinib but not erlotinib decreased EGFR mutant NSCLC PC9HRG cell proliferation in vitro and in mouse xenografts. Afatinib inhibited phosphorylation of the cell signaling pathway proteins HER3, EGFR, HER2, and HER4, likely by prevention of trans-phosphorylation as HER3 kinase activity is inadequate for auto-phosphorylation. Afatinib, unlike erlotinib, inhibited AKT activation, resulting in elevated apoptosis in PC9HRG cells. Clinically, a subpopulation of 33 patients with EGFR mutations and NSCLC who had received first generation EGFR-TKIs exhibited elevated plasma heregulin levels compared to healthy volunteers; one of these achieved a response with afatinib therapy despite having previously developed erlotinib resistance. Afatinib can overcome heregulin-mediated resistance to erlotinib in EGFR mutant NSCLC. Further studies are necessary to determine whether heregulin can predict afatinib efficacy after development offirst generation EGFR-TKI resistance.

Abstract 5311: Studies of co-expression of cancer stem cell markers and growth factor receptors in human ovarian cancer cells and their responses to treatment with various tyrosine kinase inhibitors

Abstract Ovarian cancer remains one of the most lethal types of gynaecological cancer. There is an urgent need for the identification of biomarkers for use in the early detection of such cancers and for predicting the response to therapy. Here, we investigated the sensitivity in vitro of a panel of human ovarian cancer cell lines to treatment with several small molecules tyrosine kinase inhibitors (TKIs) including reversible EGFR (Gefitinib), reversible dual EGFR/HER-2 (Lapatinib), reversible pan-ErbB family (Sapatinib), irreversible dual EGFR/HER-2 (Canertinib, Neratinib), the irreversible ErbB family blocker (Afatinib), as well as v-abl/src/C-kit (Dasatinib) and v-abl/C-kit/PDGFR TKI (Imatinib). We also determined the expression of putative ovarian cancer stem cell (CSC) markers (e.g. CD24, CD44, CD117), P-Glycoprotein (Pgp), and HER family members on this panel of cancer cells by FACS analysis and whether there was any correlation between the expression of these biomarkers and response to treatment with these agents. Of the CSC markers, the expression of CD24 was high in five of the ten ovarian cancer cell lines examined, and with MFI values ranging from of 450 (SKOV3) to 24 (SW626). In contrast, the CD24 negative ES2 cells had the highest level of expression of CD44 (MFI = 977). The MFI values for other CD44 positive tumours ranged from 139.51 (PA1) to 46.38 (Caov3). All ovarian cancer cells used in this study were negative for the expression of both CD117 and Pgp and overexpression of HER-2 (MFI = 368) was only detected in the EGFR positive SKOV3 cells. Of the pan-HER family TKIs, afatinib inhibited the growth of ovarian cancer cells with IC50 values ranging from 83nM (Caov3) to 1.40μM (ES2) and sapatinib inhibited the growth with IC50 values ranging from 24nM (Caov3) to 4.41μM (PA1) respectively. Of the two irreversible dual EGFR/HER-2 TKIs, neratinib was more effective than canertinib at inhibiting the growth of ovarian cancer cells. In contrast, Gefitinib and Lapatinib were the most effective drugs at inhibiting the growth of CaoV3 and SKOV3 cells, with IC50 values of 1.8nM and 1.4nM respectively. Interestingly, the majority of ovarian cancer cells were highly sensitive to treatment with dasatinib with IC50 values of below 3.0nM but were relatively resistant to treatment with imatinib (i.e. IC50&amp;gt;10μM). With the exceptions of correlation between the CD24/HER-2 co-expression and response to gefitinib and lapatinib, there was no statistically significant correlation between the expression of CD24, CD44, EGFR, HER-2 or other combinations of these markers and response to other agents. Our results support the need for further investigation of the predictive value of CD24/HER-2 co-expression, and the therapeutic potential of dasatinib when used in combination with the HER inhibitors in ovarian cancer. Citation Format: Soozana Puvanenthiran, Sharadah essapen, Alan Michael Seddon, Helmout Modjtahedi. Studies of co-expression of cancer stem cell markers and growth factor receptors in human ovarian cancer cells and their responses to treatment with various tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5311. doi:10.1158/1538-7445.AM2015-5311