Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer

<div><p>ROCK, or Rho-associated coiled coil-containing protein kinase, is a member of the AGC kinase family and has been shown to play a role in cell migration, ECM synthesis, stress-fiber assembly, and cell contraction. Increased ROCK expression has been reported in multiple pathological conditions, including cancer. Here, we report increased expression of ROCK 1 in pancreatic tumor epithelial cells as well as in cancer associated fibroblasts (CAF). In our analysis, 62% of tumor samples exhibited ≥2+ in staining intensity by IHC analysis, versus 40% of adjacent normal tissue samples (P<0.0001). Thus, we hypothesized that ROCKs may play a significant role in pancreatic cancer progression, and may serve as a suitable target for treatment. We report a low frequency (4/34) amplification of the ROCK1 gene locus at chromosome 18q11.1 in pancreatic ductal adenocarcinoma (PDAC) patient tissue samples by aCGH analysis. Inhibition of ROCK kinase activity by a small molecule inhibitor (fasudil) resulted in moderate (IC₅₀s of 6–71 μM) inhibition of PDAC cell proliferation, migration, and activation of co-cultured stellate cells. In the KPC mouse model for pancreatic cancer, fasudil decreased tumor collagen deposition. This translated to an enhanced overall survival of the mice and an increase in gemcitabine uptake. Though fasudil may target both the tumor epithelial cells and the CAFs, our findings are consistent with the hypothesis that inhibition of tumor stroma enhances drug penetration and efficacy in PDAC. Overall, our data suggests that ROCK1 may serve as a potential therapeutic target to enhance current treatment regimens for pancreatic cancer.</p></div

Effects of the ROCK1 inhibition on pancreatic cancer cells and cancer-associated fibroblasts.

<p>A) Fasudil dose response curves in pancreatic cancer cells treated for 72 hours. B) Tumor cell migration in ROCK1 siRNA treated cells. C) Fluorescence microscopic analysis of fasudil treated, co-cultured pancreatic cancer cells and cancer-associated fibroblasts. Cells were treated with fasudil for 48 hours and then were stained for α-SMA (red), Collagen I (green), and DNA (blue). D) Fluorescence microscopic analysis of fasudil treated CAFs. E) Fasudil treated, mono- and co-cultured pancreatic cancer cells and cancer-associated fibroblasts were harvested and analyzed by immunoblotting (dot blot) for Collagen I expression under non-denaturing conditions.</p

Effects of gemcitabine and fasudil treatment in the KPC model for PDAC.

<p>A) Pancreatic tissues harvested and stained for CD31 were analyzed for positive staining per 20x field of view. B) Mouse liver sections were cut at multiple depths to assess the presence or absence of metastatic lesions in vehicle, gemcitabine, and gemcitabine plus fasudil treated tissues. Fasudil also enhanced the tumor growth inhibitory activity of gemcitabine in KPC mice. Three-dimensional volume measurements were acquired by ultrasonography of tumor bearing KPC mice before and after one (C) or two (D) 12-day treatment cycles. Percentage change is shown from baseline. IHC analysis of the proliferation marker Ki67 (E) and apoptosis marker cleaved-caspase 3 (CC3) (F) in epithelial tumor cells are also shown for mice treated in the various treatment cohorts. Fasudil enhanced survival of tumor bearing KPC mice. G) Kaplan-Meier curves of KPC mice treated with vehicle, gemcitabine, or the combination of gemcitabine plus fasudil. The combination treatment yields a significant improvement in overall survival compared to gemcitabine only group (Log-rank P value = 0.038). H) Pancreatic tissue from mice treated for three days with either vehicle or fasudil prior to a gemcitabine injection were analyzed for gemcitabine monophosphate concentrations in the tumor tissues. * P < 0.05. ** P < 0.01.</p

Effects of fasudil treatment on tumor stroma in KPC mice.

<p>A) Pancreatic tumor tissues from vehicle, gemcitabine, or combination of gemcitabine plus fasudil treated KPC mice were harvested and stained for various stromal markers. Representative images are shown of H&E staining, α-SMA, Desmin, CD31, Collagen I, and Movat's pentachrome staining.</p

Summary of immunohistochemical staining of ROCK1 in PDAC tissue microarrays

<p>Summary of immunohistochemical staining of ROCK1 in PDAC tissue microarrays</p

Knockdown of ROCK1 by siRNA in pancreatic cancer cell inhibits cell proliferation.

<p>A) ROCK1 was detected in pancreatic cancer cell lines (PANC-1, Mia PaCa-2, SU.86.86, BxPC3, AsPC-1, and HS766T), the immortalized normal pancreatic ductal epithelial cell line (HPDE6), and the cancer associated fibroblasts (CW-1) by Western blotting. B) Western blotting analysis of ROCK1 knockdown by siRNA over the course of 72 hours. (C) Western blotting analysis of ROCK1 knockdown by siRNA (72 hour treatment) in two cell lines, SU.86.86 and PANC-1. (D) Growth curves of pancreatic cancer cells (PANC-1 and SU.86.86) treated with siRNA to ROCK1. * P < 0.001 (compared to the untreated control). UT, untreated; tR, transfection reagent only; NT, non-targeting; ASD, cell death (positive) control; R1, ROCK1 siRNA1; R2, ROCK1 siRNA 2.</p

Pharmacological targeting of TFIIH suppresses KRAS mutant pancreatic ductal adenocarcinoma and synergizes with TRAIL

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (Triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TNF-related apoptosis inducing ligand (TRAIL), effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy with direct translational implications for current clinical trials on metastatic pancreatic cancer patients

TP-0903 is active in models of drug-resistant acute myeloid leukemia

Effective treatment for AML is challenging due to the presence of clonal heterogeneity and the evolution of polyclonal drug resistance. Here, we report that TP-0903 has potent activity against protein kinases related to STAT, AKT, and ERK signaling, as well as cell cycle regulators in biochemical and cellular assays. In vitro and in vivo, TP-0903 was active in multiple models of drug-resistant FLT3 mutant AML, including those involving the F691L gatekeeper mutation and bone marrow microenvironment–mediated factors. Furthermore, TP-0903 demonstrated preclinical activity in AML models with FLT3-ITD and common co-occurring mutations in IDH2 and NRAS genes. We also showed that TP-0903 had ex vivo activity in primary AML cells with recurrent mutations including MLL-PTD, ASXL1, SRSF2, and WT1, which are associated with poor prognosis or promote clinical resistance to AML-directed therapies. Our preclinical studies demonstrate that TP-0903 is a multikinase inhibitor with potent activity against multiple drug-resistant models of AML that will have an immediate clinical impact in a heterogeneous disease like AML

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo