Tolosa, Ezequiel J.

Abstract Immunoglobulin (Ig) secretion by terminally differentiated B-cells is important in the immune response to pathogens. Its over production is a defining characteristic of many B-cell malignancies, including Waldenström Macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Thus, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for these diseases. Here, we define a novel pathway involving the oncogenic transcription factor, GLI2, modulating IgM secretion via regulation of IL-6 receptor alpha subunit (IL6Rα) expression in WM cells. We have previously identified components of the Hedgehog (HH)-GLI pathway as modulators of Ig secretion. Pharmacological (GANT61) and genetic (shRNA) inhibition of GLI proteins resulted in no significant effect on cell proliferation or viability, while there was a significant reduction in IgM secretion (60%) in BCWM.1 (p<0.0001), (55%) in MWCL-1 (p<0.0001) and (50%) in RPCI-WM1 (p<0.0001) cells. Interestingly, this occurred independent of HH signaling as manipulation of HH had no effect on IgM secretion, cell growth or viability. Screening for a mechanism identified IL6Rα as a downstream direct target of GLI activity. GANT61 treatment significantly reduces IL6Rα promoter activity in BCWM.1 (p=0.0002), MWCL-1 (P=0.0003) and RPCI-WM1 (p<0.0001) cells. GLI2 knockdown using two independent shGLI2 constructs resulted in reduced IL6Rα mRNA expression (p<0.0001) determined by qPCR, and IL6Rα protein expression examined by FACS analysis. Similarly, GLI2 knockdown diminished IL6Rα promoter activity in BCWM.1 (p=0.0218), MWCL-1 (p=0.0008) and RPCI-WM1 (p=0.0002) cells. Using bioinformatics analysis, we identified three candidate GLI2 binding sites in the IL6Rα promoter region. Using chromatin immunoprecipitation assay (ChIP) followed by qPCR, we confirmed that GLI2 directly binds to the IL6Rα promoter region (-1294 / -1065). Finally, we were able to rescue the reduction in IgM secretion in GLI2 knockdown group by overexpressing IL6Rα (p=0.0142). Taken together, these data reveal a novel mechanism where GLI2 modulates IgM secretion in WM through the regulation of IL6Rα expression. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Autophagy has been shown to be a key cellular event controlling tumor growth in different neoplasms including hepatocellular carcinoma (HCC). Although this biological role of autophagy has been clearly established, the mechanism underlying its regulation remains elusive. Here, we demonstrate a role of sulfatase 2 (SULF2), a 6‐O‐endosulfatase modulating various growth factors and cytokine‐related signaling pathways controlling tumor cell proliferation and survival, in the regulation of autophagy in HCC cells. SULF2 increased autophagosome formation, shown by increased LC3B‐II protein and green fluorescent protein–LC3 puncta. Increased fusion between autophagosomes and lysosomes/lysosomal enzymes, higher expression of lysosomal membrane protein, and an increase in autolysosomes were also shown by western blot, immunofluorescence, and electron microscopy of SULF2‐expressing cells, indicating enhanced autophagic flux. In contrast, RNA‐interference silencing of SULF2 in Huh7 cells induced lysosomal membrane permeabilization with diffuse cytosolic staining of cathepsin D and punctate staining of galectin‐3. Analysis of the mechanism showed that inhibition of lysosome‐associated protein transmembrane 4 beta (LAPTM4B), a gene induced by SULF2, resulted in decreased autophagosome formation, decreased fusion between autophagosomes and lysosomes, and increased lysosomal membrane permeabilization. Interestingly, down‐regulation of LAPTM4B also phenocopies the knockdown of SULF2, significantly reducing cell viability and colony formation. Conclusion: Our results demonstrate a role for SULF2 in the regulation of autophagic flux that is mediated through LAPTM4B induction in HCC cells, and provide a foundation for future translational efforts targeting autophagy in liver malignancies.

3101 Background: PDO is a promising translational tool that recapitulates the biology and drug response of donor cancer patient. However, an unmet need is to have PDO drug-screening data available for treatment decision making in clinic. We conducted a pilot study to determine whether PDO testing results will be available at critical treatment decision points in metastatic GI cancer patients. Methods: Metastatic GI cancer patients undergoing core-needle biopsy were eligible. Tumor cells isolated from ≤4 fresh biopsy tissues were grown in a Matrigel-based culture. PDO response to anti-cancer drugs were evaluated; and when available, correlated with donors’ clinical response to the same agent(s). PDO response was defined as IC50 < 0.1 × published Cmax of the drug clinically; stable as IC50 between 0.1 to 10 × Cmax. Radiographic response was per RECIST criteria. Results: We enrolled 27 refractory metastatic GI cancer patients (9 colorectal [CRC], 9 pancreas, and 9 biliary tract). Median lines of therapy were 4, 2, and 2; the success rate of organoid establishment was 89%, 44%, and 55%, respectively. The median time from biopsy to availability of drug-testing data was 64 days (range: 24 to 93 days). The median time from biopsy to next CT re-staging in donors was 64 days. The established PDOs shared histological and genomic features with donor clinical tissue. PDO and clinical responses to the same agent(s) were correlated in 2 CRC donors including (1) BRAFV600E-mutated PDO responded to vemurafenib + panitumumab, as did the donor who had partial response (PDO drug-testing data were available 55 days post-biopsy, 23 days prior to restaging scan); (2) KRAS/FGF-dual amplified PDO had stable disease status to regorafenib, as did the biopsied lesion from the donor (73 days post-biopsy, 5 days post-scan). Conclusions: We showed the feasibility of completing PDO drug sensitivity testing in metastatic GI cancer patients within a short time that could impact clinical decision making, particularly in CRC. PDO drug response showed correlation with clinical response. With further refinement, PDO can be a powerful tool for personalizing cancer therapy in metastatic GI cancer patients.

Objective Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis. Design DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo. Results We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer. KMT2D is transcriptionally repressed in human pancreatic tumours through DNA methylation. Clinically, lower levels of this methyltransferase associate with poor prognosis and significant weight alterations. RNAi-based genetic inactivation of KMT2D promotes tumour growth and results in loss of H3K4me3 mark. In addition, KMT2D inhibition increases aerobic glycolysis and alters the lipidomic profiles of pancreatic cancer cells. Further analysis of this phenomenon identified the glucose transporter SLC2A3 as a mediator of KMT2D-induced changes in cellular, metabolic and proliferative rates. Conclusion Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.

Abstract Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvβ3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFβ1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Cancer Res; 77(3); 632–45. ©2016 AACR.

Abstract Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.