Citations

Abstract Purpose: Waldenström's macroglobulinemia is an incurable lymphoproliferative disorder driven by an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), which activates downstream NF-κB signaling through the Myddosome. As this pathway depends in part on activity of interleukin-1 receptor-associated kinases (IRAKs)-1 and -4, we sought to evaluate the potential of the IRAK1/4 inhibitor R191 in preclinical models. Experimental Design: Patient-derived cell lines and primary samples were used in both in vitro and in vivo experiments to model Waldenström's macroglobulinemia and its response to IRAK1/4 inhibitors. Results: R191 induced a dose- and time-dependent reduction in viability of BCWM.1 and MWCL-1 Waldenström's cell lines, and suppressed activation of IRAK1/4. This was associated with cell-cycle arrest at G0–G1, reduced levels of cyclin-dependent kinases 4 and 6, and induction of apoptosis in cell lines and primary patient samples. Further downstream, R191 exposure led to reduced activation of NF-κB, and of protein kinase B/Akt/mammalian target of rapamycin signaling, whereas expression of a constitutively active Akt mutant induced R191 resistance. Gene expression profiling and gene set enrichment analysis revealed a signature consistent with inhibition of c-Myc and activation of the endoplasmic reticulum stress response. In both subcutaneous and systemic murine models of Waldenström's, R191 showed antitumor activity. Finally, the activity of R191 was enhanced when it was combined with novel chemotherapeutics such as bortezomib, afuresertib, and ibrutinib. Conclusions: Taken together, these data support the translation of R191 as an approach to target IRAK1/4 to the clinic for patients with Waldenström's macroglobulinemia.

Abstract Background: High Grade Serous Ovarian Cancer (HGSOC) is a highly aggressive form of ovarian cancer accounting for up to 70% of all cases. The Cancer Genome Atlas (TCGA) has further defined 4 molecular subtypes within HGSOC: differentiated (DIF), immunoreactive (IMM), mesenchymal (MES), and proliferative (PRO). The MES subtype is associated with the worst prognosis and the least likely to be completely resectable when compared with other subtypes. A recent study suggested the existence of a 5-th, so-called anti-mesenchymal (ANM), subtype which is characterized by down-regulation of genes typically upregulated in the MES subtype. Importantly, previous molecular subtype studies have been performed using tumor samples consisting of both cancer cells and stroma. However, the origin of MES biomarkers in OC is still largely unknown and several studies suggest that many of the MES biomarkers could arise from cancer-associated stroma rather than cancer cells. Methods: Specimens from 20 patients (MES subtype, n=15; ANM subtype, n=5) diagnosed with HGSOC between 2010 and 2013 were obtained from Pathology Research Core in Mayo Clinic. A total of 60 FFPE blocks from 1 primary tumor and 2 metastatic sites of each patient were used to make the slides. Five MES-subtype biomarkers, including ACTA2, FAP, POSTN, COL5A1, and VCAN, were selected for immunohistochemistry (IHC) to determine the protein localization and intensities. Two researchers scored tumor sections for intensity as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), and for localization (stroma/tumor, cytoplasm/nuclear). Statistical analysis was performed using GraphPad Prism 7 software and statistical differences were evaluated using t-test. Results: All 5 proteins evaluated exclusively localized in stroma rather than cancer cells (p<0.001 for all 5 proteins). Only FAP showed significantly different staining intensity between MES and ANM subtype of OC patient samples (p= 0.0114). When comparing primary vs. metastatic sites, only COL5A1 showed significant difference (p=0.019). Conclusion: We report that 5 biomarkers traditionally associated with the MES subtype HGSOC arise from cancer-associated stroma instead of cancer cells. Considering the potential roles of these proteins in mediating HGSOC behavior, it may suggest “stroma targeting” as an alternative therapy to “cancer targeting” to improve OC patient outcomes. Although all 5 genes were reported as MES biomarkers in mRNA-based analysis, we found that FAP was the only protein differentially expressed between MES and ANM subtype suggesting it is a better characteristic biomarker for MES subtype. COL5A1 showed significance in staining between primary and metastatic cancer which may indicates the potential role of COL5A1 in metastasis in OC. Citation Format: Qing Zhang, Kimberly R. Kalli, Chen Wang, William Cliby. The mesenchymal subtype biomarkers arise from stroma rather than cancer cells in ovarian cancer [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 4491.

Abstract When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O6-methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the “fitness” of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the “fitness” of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy. Citation Format: Valya Ramakrishnan, Johnny Akers, Thien Nguyen, Aaron Wang, Bandita Adhikari, Brian Hirshman, Jie Li, Jann Sarkaria, Wei Hua, Mao Ying, Masayuki Nitta, Tao Jiang, Bob Carter, Clark C. Chen. miR-181d degradation mediated genetic heterogeneity and acquired resistance [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 1956.

Abstract The liver is the most common site of metastasis in pancreatic ductal adenocarcinoma (PDAC). This metastatic tropism is dependent, at least in part, on the formation of a “pro-metastatic” niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that direct the formation of this niche remain poorly understood. We show using the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) model of PDAC that pancreatic tumor development enhances the susceptibility of the liver to metastatic seeding by inducing recruitment of F4/80+ and Ly6G+ myeloid cells and fibrosis within the liver. 3' mRNA sequencing (QuantSeq) on RNA isolated from the liver of KPC mice versus control PC mice revealed that the liver produces a specific set of myeloid chemoattractants, particularly serum amyloid A1 and A2 (SAA1/2), early during PDAC development. In addition, gene set enrichment analysis (GSEA) on genes upregulated in the liver of KPC mice demonstrated a significant enrichment of the interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway. Consistent with this finding, phosphorylation of STAT3 was detected in 20-30% of F4/80+ myeloid cells and 80-90% of hepatocytes. A requirement for IL-6/STAT3 signaling in the formation of a pro-metastatic niche was determined by comparing the metastatic potential of wild type mice, Il-6 knockout (Il-6-/-) mice, and mice treated with anti-IL-6 receptor (IL-6R) antibody after orthotopic implantation of KPC-derived PDAC cells. Compared to wild type mice, the liver of Il-6-/- mice and mice treated with anti-IL-6R antibody was less susceptible to metastatic seeding and showed significantly less accumulation of myeloid cells, fibrosis, and production of SAA1/2 in the liver. We obtained similar results with mice that lack Stat3 specifically in hepatocytes (Stat3flox/flox Alb-Cre), demonstrating that IL-6/STAT3 signaling in hepatocytes is necessary for the formation of a pro-metastatic niche in the liver. Further, using Saa1/2 double knockout (Saa-/-) mice, we found that SAA1/2 production by hepatocytes was required for formation of the pro-metastatic niche in the liver and increased susceptibility to metastatic seeding. Patients with a history of liver metastasis also showed higher levels of SAA1/2 in the plasma compared to normal donors, and SAA overexpression was detected in hepatocytes in liver biopsy samples collected from PDAC patients. Collectively, our study reveals a novel role for hepatocytes in directing the formation of a pro-metastatic niche in the liver during PDAC development and identifies IL-6/STAT3/SAA1/2 signaling as a promising therapeutic target for prevention of metastasis in PDAC. Citation Format: Jae W. Lee, Stacy K. Thomas, Chad A. Komar, Whitney L. Gladney, Xia Hua, Dong Xin, Abraham Shaked, Mitesh J. Borad, Ramesh K. Ramanathan, Ailing Ji, Nancy R. Webb, Maria C. de Beer, Frederick C. de Beer, Paige M. Porrett, Gregory L. Beatty. IL-6/STAT3 activation in hepatocytes drives pro-metastatic niche formation in the liver [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 1102.

Background: Since healing of anterior cruciate ligament (ACL) grafts occurs by formation of a fibrovascular scar-tissue interface rather than by reformation of the native fibrocartilage transition zone, the purpose of our study was to examine expression of various signaling molecules and transcription factors that are known to be involved in embryologic insertion-site development following ACL reconstruction. We also aimed to characterize a murine model of ACL reconstruction to allow future study of the molecular mechanisms of healing. Methods: Seventy-nine mice underwent reconstruction of the ACL with autograft. Healing was assessed using histology in 12 mice and quantitative real-time polymerase chain reaction (qRT-PCR) gene-expression analysis in 3 mice at 1 week postoperatively (Group-1 mice) and by biomechanical analysis in 7, histological analysis in 7, immunohistochemical analysis in 5, microcomputed tomography analysis in 5, and qRT-PCR analyses in 8 at 2 weeks (Group-2 mice) and 4 weeks (Group-3 mice) postoperatively. Fifteen additional mice did not undergo surgery and were used for biomechanical (7 mice), qRT-PCR (3 mice), and immunohistochemical (5 mice) analyses to obtain baseline data for the native ACL. Results: Histological analysis demonstrated healing by formation of fibrovascular tissue at the tendon-bone interface. Immunohistochemical analysis showed a positive expression of proteins in the Indian hedgehog, Wnt, and parathyroid hormone-related protein (PTHrP) pathways. There was minimal Sox-9 expression. Gene-expression analysis showed an initial increase in markers of tissue repair and turnover, followed by a subsequent decline. Mean failure force and stiffness of the native ACL were 5.60 N and 3.44 N/mm, respectively. Mean failure force and stiffness were 1.29 N and 2.28 N/mm, respectively, in Group 2 and were 1.79 N and 2.59 N/mm, respectively, in Group 3, with 12 of 14 failures in these study groups occurring by tunnel pull-out. Conclusions: The spatial and temporal pattern of expression of signaling molecules that direct embryologic insertion-site formation was not adequate to restore the structure and composition of the native insertion site. Clinical Relevance: Development of a murine model to study ACL reconstruction will allow the use of transgenic animals to investigate the cellular, molecular, and biomechanical aspects of tendon-to-bone healing following ACL reconstruction, ultimately suggesting methods to improve healing in patients.

Abstract Background Previous genome-wide association studies (GWAS) have identified 42 loci (P < 5 × 10−8) associated with risk of colorectal cancer (CRC). Expanded consortium efforts facilitating the discovery of additional susceptibility loci may capture unexplained familial risk. Methods We conducted a GWAS in European descent CRC cases and control subjects using a discovery–replication design, followed by examination of novel findings in a multiethnic sample (cumulative n = 163 315). In the discovery stage (36 948 case subjects/30 864 control subjects), we identified genetic variants with a minor allele frequency of 1% or greater associated with risk of CRC using logistic regression followed by a fixed-effects inverse variance weighted meta-analysis. All novel independent variants reaching genome-wide statistical significance (two-sided P < 5 × 10−8) were tested for replication in separate European ancestry samples (12 952 case subjects/48 383 control subjects). Next, we examined the generalizability of discovered variants in East Asians, African Americans, and Hispanics (12 085 case subjects/22 083 control subjects). Finally, we examined the contributions of novel risk variants to familial relative risk and examined the prediction capabilities of a polygenic risk score. All statistical tests were two-sided. Results The discovery GWAS identified 11 variants associated with CRC at P < 5 × 10−8, of which nine (at 4q22.2/5p15.33/5p13.1/6p21.31/6p12.1/10q11.23/12q24.21/16q24.1/20q13.13) independently replicated at a P value of less than .05. Multiethnic follow-up supported the generalizability of discovery findings. These results demonstrated a 14.7% increase in familial relative risk explained by common risk alleles from 10.3% (95% confidence interval [CI] = 7.9% to 13.7%; known variants) to 11.9% (95% CI = 9.2% to 15.5%; known and novel variants). A polygenic risk score identified 4.3% of the population at an odds ratio for developing CRC of at least 2.0. Conclusions This study provides insight into the architecture of common genetic variation contributing to CRC etiology and improves risk prediction for individualized screening.