e15185 Background: Aberrant activation of Wnt signaling contributing to tumorigenesis is most commonly associated with CRC (90% harbor Wnt pathway mutations). SM08502, a novel, oral Wnt signaling pathway inhibitor, was evaluated in preclinical CRC models. Methods: In vitro Wnt signaling: assessed using TOPflash β-catenin/TCF reporter assay in SW480 human CRC cells. In vitro Wnt pathway gene expression: measured by qRT-PCR in SW480 and Wnt3a-stimulated cells (HEK-293T, IEC-6), and with the Nanostring Wnt pathway array (180 genes) across a panel of 16 CRC cell lines. In vitro cell proliferation: 17 CRC cell lines were used to test cell viability following treatment. In vivo antitumor activity: Oral SM08502 was tested in CRC mouse xenografts (SW480, HCT 116) and a PDX model over 20-21 days (QD, QOD). 24-hr pharmacodynamic (PD) analysis of Wnt pathway gene expression was done in SW480 tumor explants from mice following one 25 mg/kg dose. Results: SM08502 inhibited Wnt pathway signaling (EC50 = 46 nM) in SW480 cells. Wnt pathway gene expression was inhibited by SM08502 (0.3-3 µM) in Wnt3a-stimulated cells ( AXIN2, LEF1) and SW480 ( AXIN2, CTNNB1, LEF1, MYC, TCF7, TCF7L2) at 24 hrs ( P < .05 vs. vehicle) . Corresponding effects on protein expression were confirmed for all genes except CTNNB1, suggesting SM08502 acted independently of β-catenin. Nanostring array screening identified inhibition of LRP5, DVL2, BTRC, and ERBB2 by SM08502. Cell proliferation was inhibited in all 17 lines (avg. EC50 = 177 nM). In vivo, SM08502 was well tolerated and induced dose-dependent antitumor effects in xenografts and PDX models. Tumor growth inhibition for 25 mg/kg QD (max dose) was 83%, 56%, and 70% in SW480, HCT 116, and PDX, respectively. PD analysis showed significant inhibition ( P< .05 vs. vehicle) of TCF7, MYC, LRP5, DVL2, and BTRC expression 8 hrs post treatment. Conclusions: In preclinical CRC models, SM08502 was a potent inhibitor of Wnt pathway signaling and gene expression. It showed strong antitumor activity in human tumor models with activating Wnt pathway mutations. The safety, tolerability, and PK of SM08502 are being evaluated in an ongoing phase 1 study (NCT03355066).
Isolated hypoglossal nerve (CN XII) palsy is rare. Neurological complications of multiple myeloma (MM) are quite common, most often due to hyperviscosity and paraprotein-related neuropathy. Direct compression of CN XII can be caused by plasmacytoma, yet direct invasion by MM is extremely rare. We are reporting a very unusual case of a 45-year-old man who presented with an isolated right CN XII palsy. The cause revealed by MRI is stenosis of the hypoglossal canal resulting from lytic bony erosion. Despite negative serum and urine protein electrophoresis tests, the final diagnosis of oligosecretory MM was confirmed by serum-free light chain test and bone marrow biopsy. The causes and diagnosis of isolated XII nerve palsy and oligosecretory MM are discussed.
In prostate cancer, alternative splicing of mRNA and spliceosome activity are implicated in several areas of disease pathogenesis. This is exemplified by the strong association of androgen receptor splice variants with treatment resistance and poor clinical outcome in castration-resistant disease. Therefore, pharmacologic targeting of spliceosome-regulating proteins such as CLKs and serine/arginine-rich splicing factors (SRSFs) represents a novel treatment approach for prostate cancer. To evaluate the therapeutic potential of inhibiting CLK activity in prostate cancer, the association between splicing-related gene expression and survival was investigated in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data collection (N=495). Survival analysis of RNA-seq data assessed 17,879 genes to measure their association with progression-free interval (PFI). Using transcript per million as the metric for normalized gene expression, age-adjusted Cox proportional hazards regression models were performed for each gene (R v3.6.0, coxph v2.43-3). A total of 3,145 genes significantly correlated with worse prognosis (P-adj<0.10, Cox coefficient >0). CLK1 (P-adj=0.0218, HR=1.5939), CLK2 (P-adj=0.001298, HR=2.1393), and SRSF2 (P-adj=0.00167, HR=3.2917) were found to be positively associated with poorer PFI, ranking 1202, 400, and 437, respectively. Reactome pathway analysis of the significant gene set showed that mRNA splicing and processing accounted for 5 of the 19 pathways that were strongly associated with poorer PFI. An additional pathway analysis (GSEA v.3.0, MSigDB v6.2) of tumors categorized by PTEN status to assess relationship with disease severity showed that mRNA splicing (P-adj=0.0243, NES=1.7714) was enriched in PTEN-null vs. PTEN-wt tumors. Other pathways of interest, including Wnt signaling (P-adj=0.0187, NES=1.846), cell cycle (P-adj=0.0124, NES=1.974), chromatin remodeling (P-adj=0.0135, NES=1.901), DNA damage repair (P-adj=0.013974, NES=1.8934), and PTEN regulation (P-adj=0.0230, NES=1.7861), were also enriched in PTEN-null tumors. Lastly, a survival analysis within all TCGA-PRAD patients showed that low CLK1 (P=0.03) and CLK2 (P=0.0004) expression, individually, were associated with a better prognosis vs. their high-expressing counterparts. Analysis of CLK3 and CLK4 expression did not reach statistical significance. Collectively, these findings revealed an association of spliceosome activity and CLK1/2 expression with aggressive disease biology in prostate cancer. A Phase 1 study of SM08502, a novel, small-molecule pan-CLK inhibitor, in subjects with advanced solid tumors is ongoing (NCT03355066). This analysis nominates prostate cancer as a tumor type worth further exploring for the clinical activity of SM08502. Citation Format: Shawn Cho, Atish D. Choudhury, Catherine Fleener, Long Do, Carine Bossard, Heekyung Chung, Timothy J. Phalen, Steven Cha. Transcriptome analysis of TCGA prostate cancer samples identifies an association of poorer survival and aggressive disease biology with CDC-like kinase (CLK) expression and spliceosome regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3521.
BackgroundIn the synovial joint, Wnt pathway upregulation contributes to osteoarthritis (OA) by increasing osteocyte differentiation, cartilage thinning, and inflammation. SM04690, a novel small molecule, has previously demonstrated potential OA disease-modifying effects through Wnt pathway inhibition in vitro and in vivo.ObjectivesTo elucidate the novel mechanism of action for SM04690 on Wnt pathway inhibition, chondrocyte differentiation, and anti-inflammation.MethodsWnt pathway activity was measured using a cell-based TCF/LEF luciferase reporter in SW480 colon cancer cells. A kinome screen (318 kinases) was performed. The effects of SM04690 on protein phosphorylation of serine and arginine rich splicing factors (SRSF proteins), Sirt1, and FoxO1 in hMSCs, chondrocytes, and synovial fibroblasts were measured by Western blot. The effects of SM04690 and siRNA knockdown (KD) on chondrogenic and Wnt pathway gene expression were measured by NanoString gene expression panels and effects on LPS-induced inflammatory cytokines (IL-6, IL-8, TNF-α) in BEAS-2B cells were measured by qPCR and ELISA. In vivo, the pharmacodynamic effects of SM04690 were evaluated in monosodium iodoacetate injection-induced and anterior cruciate ligament transection with partial medial meniscectomy rat knee OA models in which a single intra-articular SM04690 (0.1 µg, 0.3 µg, 1.0 µg) or vehicle injection was administered. Cartilage was isolated at Day 10 and 35; phosphorylation and expression of SRSF proteins, Sirt1, FoxO1, STAT3, and NF-κB were measured by Western blot.ResultsSM04690 was a potent (EC50=11nM) inhibitor of Wnt signaling. Cdc-like kinases (CLKs) and dual-specificity tyrosine kinase (DYRK1A) were identified as molecular targets of SM04690. In hMSCs and chondrocytes, compared to DMSO, SM04690 potently inhibited CLK-mediated phosphorylation of SRSF proteins. SM04690 also inhibited DYRK1A-mediated Sirt1 and FoxO1 phosphorylation, thus increasing total and nuclear FoxO1 levels. Compared to siRNA control, DYRK1A/CLK2 dual KD increased expression of chondrogenic genes (COL2A1, ACAN, COMP, CD44 [all P<0.05]). CLK2 and DYRK1A KDs each inhibited Wnt pathway genes (AXIN2, TCF7, TCF4, LRP5, FZD6, FZD7, PITX2 [all P<0.05]) with no effects on β-catenin levels, compared to siRNA control. In synovial fibroblasts, compared to DMSO, SM04690 decreased phosphorylation of NF-kB and STAT3. In BEAS-2B cells, compared to siRNA control, DYRK1A KD inhibited inflammatory cytokine production (IL-6, IL-8, TNF-α [all P<0.05]), while DYRK1A/CLK2 dual KD enhanced anti-inflammatory effects of DYRK1A KD. In cartilage from rat OA models, compared to vehicle, SM04690 inhibited phosphorylation of SRSF proteins, Sirt1, FoxO1, and STAT3, as well as expression of NF-κB.ConclusionTo our knowledge, this is the first report of SM04690 inhibition of nuclear kinases CLK2 and DYRK1A, leading to effects on the Wnt pathway, chondrocytes, and inflammation (Figure 1). This dual mechanism of SM04690 potentially modifies OA through increased chondrocyte differentiation and function ...
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