Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or ‘priming’, improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as ‘priming’ agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease.</p

Supplementary Figures S1-S8 from Selected Alkylating Agents Can Overcome Drug Tolerance of G₀-like Tumor Cells and Eradicate BRCA1-Deficient Mammary Tumors in Mice

Figure S1: Treatment response to cisplatin spread over several days. Figure S2: Cisplatin adducts in tumor remants; GFP-marker in the BRCA1-deficient mouse model. Figure S3: EdU incorporation assay. Figure S4: Genomic profile and clonogenic assays of BRCA1-deficient cell lines. Figure S5: Determination of IC50 in KB1P-B11 and -G3; Cell cycle dependent cell death of KB1P-B11-FUCCI. Figure S6: Drug response to nimustine, melphalan, bendamustine or temozolomide. Figure S7: High dose treatment of mice with carboplatin or thiotepa. Figure S8: Combination or single treatment with cyclophosphamide, carboplatin or thiotepa.</p

Supplementary Methods from Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Detailed methods of: EGFR immunohistochemistry of PDAC tissue microarrays Patient derived xenografts (PDXs) and cell lines (PDCLs) generation Cell culture Treatments in vitro Histological and immunoblotting analyses of PDXs post-treatment</p

Supplementary Figure 2 from Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Supplementary Figure 2: Maximum tolerated doses of gemcitabine, Chk1i and EGFR-directed RIT in vivo. (A) Balb/c nude mice bearing PANC-1 subcutaneous xenografts (60 mm3 in volume) were treated with 50 mg/kg or 100 mg/kg of gemcitabine on days 1, 4, 7 and 10 administered intravenously combined with 15 mg/kg Chk1i, administered as two doses per day (7.5 mg/kg per dose) on days 1, 4, 7 and 10 at 3 hours before and after gemcitabine administration. Mice were treated with gemcitabine and Chk1i combinations (gem + Chk1i) alone or in combination with 6 or 9 MBq/20g (300 or 450 MBq/kg) of 177Lu-anti-EGFR mAb. Mice were monitored for 14 days to determine acute toxicities as judged by weight, posture, movement, eating and drinking. Tumor volume was also monitored during this short time as an indication of efficacy. (B) The tumor growth curves were used to measure the growth rate (k = day-1) from exponential growth equations and shown in the bar graph in panel (5 mice per group, error bar is the standard error of the mean, SEM). *** p ® Prism comparing treatments involving anti-EGFR RIT and those not involving this RIT (p<0.001). No statistical differences were observed amongst treatments involving the anti-EGFR RIT.</p

Supplementary Figure 4 from Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Supplementary Figure 4: Efficacy of anti-EGFR directed RIT in combination therapy in vivo against PDAC model from the BxPC-3 ATCCTM cell lines. BxPC-3 cells expressing luciferase were used to inoculate balb/c nude mice subcutaneously at 4 weeks of age. Treatments were initiated when tumors reached 60 mm3 as outlined in diagram in A and in Figure 2. Single agent treatments included: EGFR control: 50 μg of unlabeled anti-EGFR mAb; Chk1i: 15 mg/kg of the Chk1 inhibitor PF-477736 administered as two 7.5 mg/kg injections per day subcutaneously on days 1, 4, 7 and 10; Gem: 50 mg/kg of gemcitabine administered intravenously on days 1, 4, 7 and 10; RIT: 177Lu-anti-EGFR mAb (50 μg with 6 MBq radioactivity per 20 g mouse) injected intravenously on day 2 only. For combinations, treatments were performed as described for the single agents where Chk1i was administered 3 hours before and after gemcitabine on days 1, 4, 7 and 10 and RIT was administered on day 2 only. (B) Tumor growth curves presented as the change in tumor volume compared to day 0 (% change +SEM, n = 5 mice per treatment group). (C) Representative images of live bioluminescence imaging using luciferin performed on day 1 (prior to treatment) and day 14 (96 hours after treatments). (D) Quantification and representative images of live bioluminescence imaging using caspase-3/7 substrate (Z-DEVD-Luciferase) performed on day 7 after treatment initiation. Data shown is the average of caspase-3/7 activation (+SEM, n = 5 per treatment group). *** p ® Prism.</p

Supplementary Figure 3 from Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Supplementary Figure 3: EGFR expression in the PANC-1 and BxPC-3. Cell cultures were used for standard immunoblot and flow cytometry analysis for EGFR expression. The anti-EGFR mAb clone 225 was used for staining and anti-mouse IgG antibody conjugated with HRP was used for immunoblot or conjugated with Alexa488 for flow cytometry. Anti-tubulin was used to confirm equal loading for immunoblot. Both PDAC cell lines express EGFR.</p

Supplementary Figure 1 from Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Supplementary Figure 1: Sensitization of PANC-1 cells to gemcitabine and EGFR-directed RIT by Chk1 inhibition. (A) Adherent cultures of the PDAC PANC-1 cell line were treated in the absence or presence of escalating doses of gemcitabine, in the absence of presence of Chk1i alone (PF-477736, 180 nM) or the combinations. Chk1i was added either 16 hours after gemcitabine or concurrently. Cells were collected 24-96 hours after treatment for standard cell cycle analysis by DNA content using FACS. (B) PANC-1 cells were treated with escalating concentrations of 177Lu-anti-EGFR mAb (RIT) to achieve the specified radiation doses (0-4 Gy) over 72 hours of incubation. RIT was performed alone or in combination with Chk1i (180 nM) and then clonogenic survival was determined by standard assays. Chk1i alone at 180 nM did not have any effect on clonogenic survival (data not shown). (C) PANC-1 cells were left untreated (vehicle control, data not shown) or incubated with anti-EGFR mAb (unlabeled, control which was not different from the vehicle control), 177Lu-DOTA, 177Lu-labelled mAb with irrelevant specificity (Sal5, raised against Salmonella antigen) or 177Lu-anti-EGFR mAb (2 Gy over 72 hours). Cells were washed 3 hours after incubation to remove unbound material and left untreated or treated with Chk1i alone (180 nM), gemcitabine alone (40 ng/mL) or the combination (Chk1i+gemcitabine) before standard clonogenic survival assays.</p

Additional file 5 of Systematic functional identification of cancer multi-drug resistance genes

Additional file 5: Table S4. This table contains a list of gene hits and scores after MAGeCK analysis

Additional file 3 of Systematic functional identification of cancer multi-drug resistance genes

Additional file 3: Table S2. This table contains sgRNA level data after MAGeCK analysis

Image_1_Oral Squamous Cell Carcinoma in Young Patients Show Higher Rates of EGFR Amplification: Implications for Novel Personalized Therapy.jpeg

There is an increasing worldwide incidence of patients under 50 years of age presenting with oral squamous cell carcinoma (OSCC). The molecular mechanisms driving disease in this emerging cohort remain unclear, limiting impactful treatment options for these patients. To identify common clinically actionable targets in this cohort, we used whole genome and transcriptomic sequencing of OSCC patient samples from 26 individuals under 50 years of age. These molecular profiles were compared with those of OSCC patients over 50 years of age (n=11) available from TCGA. We show for the first time that a molecular signature comprising of EGFR amplification and increased EGFR RNA abundance is specific to the young subset of OSCC patients. Furthermore, through functional assays using patient tumor-derived cell lines, we reveal that this EGFR amplification results in increased activity of the EGFR pathway. Using a panel of clinically relevant EGFR inhibitors we determine that an EGFR-amplified patient-derived cell line is responsive to EGFR inhibition, suggesting EGFR amplification represents a valid therapeutic target in this subset of OSCC patients. In particular, we demonstrate sensitivity to the second-generation EGFR tyrosine kinase inhibitor afatinib, which offers a new and promising therapeutic avenue versus current EGFR-targeting approaches. We propose that testing for EGFR amplification could easily be integrated into current diagnostic workflows and such measures could lead to more personalized treatment approaches and improved outcomes for this younger cohort of OSCC patients.</p