B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.

RNAi screening of the kinome with cytarabine in leukemias.

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines.

BACKGROUND: YB-1 is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. Recent data have shown that YB-1 is also overexpress in colorectal cancer. In this study, we tested the hypothesis that YB-1 also confers oxaliplatin resistance in colorectal adenocarcinomas. RESULTS: We show for the first time that transfection of YB-1 cDNA confers oxaliplatin resistance in two colorectal cancer cell lines (SW480 and HT29 cell lines). Furthermore, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. A tagged YB-1 construct was used to identify proteins interacting directly to YB-1 in such cells. We then focused on proteins that are potentially involved in colorectal cancer progression based on the Oncomine microarray database. Genes encoding for these YB-1 interactors were also examined in the public NCBI comparative genomic hybridization database to determine whether these genes are localized to regions of chromosomes rearranged in colorectal cancer tissues. From these analyses, we obtained a list of proteins interacting with YB-1 and potentially involved in oxaliplatin resistance. Oxaliplatin dose response curves of SW480 and HT29 colorectal cancer cell lines transfected with several siRNAs corresponding to each of these YB-1 interactors were obtained to identify proteins significantly affecting oxaliplatin sensitivity upon gene silencing. Only the depletion of either NONO or RALY sensitized both colorectal cancer cell lines to oxaliplatin. Furthermore, depletion of NONO or RALY sensitized otherwise oxaliplatin resistant overexpressing YB-1 SW480 or HT29 cells. CONCLUSION: These results suggest knocking down NONO or RALY significant counteracts oxaliplatin resistance in colorectal cancers overexpressing the YB-1 protein.

An integrative approach to identify YB-1-interacting proteins required for cisplatin resistance in MCF7 and MDA-MB-231 breast cancer cells.

The Y-box binding protein 1 (YB-1) is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. The exact mechanism by which YB-1 confers cisplatin resistance is unknown. The aim of the present study was to identify, using mass spectrometry, proteins that interact with YB-1 that are important for cisplatin resistance in two breast cancer cell lines, namely MCF7 and MDA-MB-231. A tagged YB-1 construct was used to identify proteins interacting directly with YB-1 in breast cancer cells. We then focused on proteins that are potentially involved in breast cancer progression based on the ONCOMINE public microarray database. Genes encoding for these YB-1-interacting proteins were examined in the public NCBI comparative genomic hybridization database to determine whether they are localized to regions of chromosomes that are rearranged in breast cancer tissues. From these analyses, we generated a list of proteins potentially involved in cisplatin resistance. Cisplatin dose-response curves were constructed in MCF7 and MDA-MB-231 transfected with four siRNA corresponding to each of these YB-1 interactors to identify proteins significantly affecting cisplatin sensitivity upon gene silencing. Depletion of only the X-linked ribosomal protein S4 (RPS4X) resulted in consistent resistance to cisplatin in both cell lines with at least three different siRNA sequences against RPS4X. Further analyses indicated that the knock down of RPS4X decreased DNA synthesis, induced cisplatin resistance, and is equivalent to the overexpression of YB-1 in both MCF7 and MDA-MB-231 cells. These results suggest that the RPS4X/YB-1 complex is a significant potential target to counteract cisplatin resistance in breast cancer.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

High-throughput siRNA screening as a method of perturbation of biological systems and identification of targeted pathways coupled with compound screening.

High-throughput RNA interference (HT-RNAi) is a powerful research tool for parallel, 'genome-wide', targeted knockdown of specific gene products. Such perturbation of gene product expression allows for the systematic query of gene function. The phenotypic results can be monitored by assaying for specific alterations in molecular and cellular endpoints, such as promoter activation, cell proliferation and survival. RNAi profiling may also be coupled with drug screening to identify molecular correlates of drug response. As with other genomic-scale data, methods of data analysis are required to handle the unique aspects of data normalization and statistical processing. In addition, novel techniques or knowledge-mining strategies are required to extract useful biological information from HT-RNAi data. Knowledge-mining strategies involve the novel application of bioinformatic tools and expert curation to provide biological context to genomic-scale data such as that generated from HT-RNAi data. Pathway-based tools, whether text-mining based or manually curated, serve an essential role in knowledge mining. These tools can be applied during all steps of HT-RNAi screen experiments including pre-screen knowledge gathering, assay development and hit confirmation and validation. Most importantly, pathway tools allow the interrogation of HT-RNAi data to identify and prioritize pathway-based biological information as a result of specific loss of gene function.

Complete regression of cutaneous metastases with systemic immune response in a patient with triple negative breast cancer receiving p53MVA vaccine with pembrolizumab.

A heavily pretreated patient with triple negative breast cancer distinguished by cutaneous metastases received p53MVA vaccine in combination with pembrolizumab. Her cutaneous metastases regressed and after 2 cycles of therapy, a skin biopsy showed a complete pathological response. Systemic response was confirmed with restaging CT and bone scans. Activation of p53-specific T cell responses and elevation of multiple immune response genes in peripheral blood correlated with the rapid clinical response which lasted for 6 months after the initiation of combined therapy.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to Aurora kinase inhibitors.

Aurora kinases are a family of mitotic kinases that play important roles in the tumorigenesis of a variety of cancers including pancreatic cancer. A number of Aurora kinase inhibitors (AKIs) are currently being tested in preclinical and clinical settings as anti-cancer therapies. However, the antitumor activity of AKIs in clinical trials has been modest. In order to improve the antitumor activity of AKIs in pancreatic cancer, we utilized a kinome focused RNAi screen to identify genes that, when silenced, would sensitize pancreatic cancer cells to AKI treatment. A total of 17 kinase genes were identified and confirmed as positive hits. One of the hits was the platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), which has been shown to be overexpressed in pancreatic cancer cells and tumor tissues. Imatinib, a PDGFR inhibitor, significantly enhanced the anti-proliferative effect of ZM447439, an Aurora B specific inhibitor, and PHA-739358, a pan-Aurora kinase inhibitor. Further studies showed that imatinib augmented the induction of G2/M cell cycle arrest and apoptosis by PHA-739358. These findings indicate that PDGFRA is a potential mediator of AKI sensitivity in pancreatic cancer cells.