R-spondin Fusion Research Articles

517MO Phase I study of the porcupine (PORCN) inhibitor RXC004 in patients with advanced solid tumours

Dysregulated Wnt signalling drives several cancers through effects on proliferation, fibrosis and immune evasion. RXC004 is a novel small molecule inhibitor of the protein-serine O-palmitoyltransferase, PORCN, which is required for post-translational modification of Wnt ligands and downstream signaling. RXC004 has potential for monotherapy efficacy in Wnt pathway activated tumours: ∼5% pancreatic and ∼ 8% microsatellite stable colorectal (CRC) cancers with RNF-43 mutations or R-Spondin fusions, and thymus and biliary tract (BTC) cancers with high Wnt ligand activity. This open label, 3+3 dose escalation study was one module of a multi-modular adaptive design protocol (NCT03447470). Following a single dose with a 7-day washout, patients received RXC004 once daily in 21-day cycles. The primary objectives were to assess safety and tolerability and define a recommended phase 2 dose of RXC004. Secondary objectives were PK and RECIST response. Pharmacodynamic markers included skin Wnt pathway (Axin2) suppression. Between 05/02/2018 and 21/06/2021, 25 patients with advanced cancers received RXC004. The first patient, who received 10mg daily, discontinued due to diarrhoea and an asymptomatic clavicle fracture was found on follow up. The half-life (T1/2) and exposure were higher than predicted from preclinical models. The study was paused and restarted at 0.5mg. Subsequently, 1mg, 1.5mg, 2mg and 3mg doses were evaluated. Patients received denosumab 120 mg s.c monthly to prevent bone adverse events [AEs]. For doses <3mg, the most common treatment related AEs were fatigue, nausea, dysgeusia, vomiting and anorexia. No grade 4/5 AEs or bone fragility events were reported. Exposure was dose proportional and median T1/2 was 14.5h. Exposures above the preclinical model IC50 were observed at all doses, as was Axin2 suppression. Five patients, all with Wnt pathway activated tumours [2 BTC, 1 Thymus, 1 mutRNF43 CRC and 1 RSPO fusion CRC] had stable disease, in one case for up to 26 weeks. In patients with unselected cancers, RXC004 was safe and tolerated at doses up to 2mg/day, supporting phase 2 development in selected patients with Wnt pathway activated tumours. Studies will open in 2021.

Exploiting the Therapeutic Interaction of WNT Pathway Activation and Asparaginase for Colorectal Cancer Therapy.

Colorectal cancer is driven by mutations that activate canonical WNT/β-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β-catenin-independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or β-catenin mutations) of GSK3, thus allowing WNT/β-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or β-catenin-mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or β-catenin-mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy. SIGNIFICANCE: Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer.See related commentary by Davidsen and Sullivan, p. 1632.This article is highlighted in the In This Issue feature, p. 1611.

Molecular landscape of colorectal cancers harboring R-spondin fusions.

3588 Background: Gene fusions involving R-spondin ( RSPO) family members have been shown to drive Wnt-dependent tumor initiation in colorectal cancer (CRC). Therapies targeting Wnt pathway are being actively investigated for tumors harboring RSPO2/3 fusions. Here we set out to characterize the molecular features of CRC with and without RSPO fusions to gain insight into potential rationale combination therapy strategies. Methods: Tumor DNA sequencing of 592 genes (NextSeq, Illumina), RNA sequencing of 53 gene fusions (ArcherDx FusionPlex) and immunohistochemistry for PD-L1 on tumor cells (SP142) were tested on CRC tumors at Caris Life Sciences, Phoenix, AZ. Molecular profiles of RSPO2/3 positive (pos) were compared with negative (neg) tumors, Fisher-Exact was used for comparative analysis. Results: A total of 1356 CRC samples were analyzed. RSPO3 and RSPO2 fusions were detected in 42 (3.1%) and 4 (0.3%) samples, respectively, including 5 fusion events not previously reported (e.g., IFNGR1-RSPO3). A female predominance was seen in RSPO fusion pos vs. neg tumors (71.7% vs 45.0%, p &lt; 0.001); no association with age or tumor sidedness was seen. RSPO2/3 fusions were mutually exclusive of MSI-high (0 vs. 5%), ERBB2 alterations (0 vs. 1% mutation, 4% amplification) and other Wnt pathway activation drivers including APC (2 vs. 75%), CTNNB1 (0 vs. 1.4%) and RNF43 (0 vs. 5.3%) mutations. Significantly higher BRAF (26 vs. 7%), RAF1 (4.5 vs. 0.4%) and SMAD4 (30 vs. 11%) mutation rates were seen in RSPO pos vs. neg tumors (p &lt; 0.05). A universal co-activation of MAPK pathway ( KRAS, NRAS or BRAF) was seen with RSPO fusions. There was a significantly elevated PD-L1 expression in RSPO3 pos tumors (14%) compared to RSPO neg (6%, p = 0.04) and APC-mutated (5%, p = 0.02) tumors that are MSS. Conclusions: This is the largest series of CRC cases harboring an RSPO rearrangement reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO fusions in CRC and suggested potential combinatorial approach to target Wnt/MAPK pathway. The immune modulatory effects specific to RSPO2/3 fusion revealed by PD-L1 expression suggest co-targeting Wnt pathway with PD1/PDL1 inhibitors in RSPO pos tumors.

A Systematic Analysis of Oncogenic Gene Fusions in Primary Colon Cancer.

Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of BRAF, NTRK3, and RET gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X-ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node-negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy. Cancer Res; 77(14); 3814-22. ©2017 AACR.

Abstract 490: Identification of oncogenic gene fusions in primary colon cancers

Abstract Introduction: Fusion genes (such as ALK-EML) are potentially attractive targets for anti-cancer treatment. In the era of personalized medicine exploring the identification, incidence, and functionality of fusion genes may contribute to effective treatment approaches. Methods: We performed a comprehensive and unbiased screening for gene fusions in a clinically well-defined prospectively collected cohort of 278 primary stage I to III colon cancers. Illumina RNA sequencing was performed using RNA from the fresh frozen samples. The STAR fusion gene detection pipeline and GATK RNA-seq variant calling were used to identify fusion genes and detect somatic genetic variations. Gene fusions were considered relevant when recurrent, when resulting in divergent expression (outlier analysis) or when functional relevance was predicted (i.e. kinase fusions). Results: 2.5% of all samples contained a relevant gene fusion. Kinase fusions were most prevalent with a frequency of 1.8%. Three BRAF-fusions were identified, two known (TRIM24-BRAF &amp; AGAP3-BRAF) and one novel fusion (DLG1-BRAF). Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The outlier analysis revealed four unique fusions (EML4-NTRK3, RRBP1-RET, USPX9-ERAS &amp; EIF3E-RSPO2). The EML4-NTRK3 fusion led to increased expression of the tyrosine kinase encoding domain exons, which is retained in the fusion transcript. Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The tumor with the RRBP1-RET fusion was pan negative for known driver mutations, as reported in literature. Mate-pair sequencing of the USPX9-ERAS fusion gene region revealed that the fusion gene was caused at the genomic level by a highly local chromothripsis event on chromosome X spanning solely the region covered by USP9X and ERAS, leading to high ERAS expression. ERAS is a constitutionally active RAS protein and normally only expressed in embryonic stem cells. Analysis of phosphorylated AKT after expression of the USP9X-ERAS fusion gene in NIH-3T3 A14 cells showed activation of AKT signaling. The occurrence of one R-spondin fusion in our set (0.4%) is low compared to the previously described 9.5%, which may be explained by difference in incidence of KRAS- or BRAF-mutations in the two cohorts, since all R-spondin fusions occurred in a tumor with a KRAS- or BRAF-mutation. All fusion genes were expressed in cell lines resulting in the activation of AKT signaling. Finally, oncogenic fusions were only found in lymph node negative tumors and were not observed in tumors with classical driver mutations in BRAF and KRAS except for the R-spondin fusion. Conclusions: We found oncogenic gene fusions including novel fusions with a frequency of 2.5%, including an USP9X-ERAS fusion with strong oncogenic activity in vitro. Although recurrent fusion genes are rare events in colorectal cancer they may represent functional drivers and provide potential novel leads for personalized therapeutic strategies. Citation Format: Robert R. Coebergh van den Braak, Wigard P. Kloosterman, Mark Pieterse, Markus van Roosmalen, Anieta M. Sieuwerts, Christina Stangl, Ronne Brunekreef, Zarina S. Lalmahomed, Salo Ooft, Anne van Galen, Marcel Smid, Armel Lefebvre, Fried J. Zwartkruis, John W. Martens, John A. Foekens, Katharina Biermann, Marco J. Koudijs, Jan N. IJzermans, Emile E. Voest. Identification of oncogenic gene fusions in primary colon cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2017-490

Novel Bispecific Domain Antibody to LRP6 Inhibits Wnt and R-spondin Ligand-Induced Wnt Signaling and Tumor Growth

Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer. This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859-68. ©2016 AACR.

Abstract 136: Refining the molecular profile of colorectal tumors to expand prevention and treatment opportunities

Abstract The completion of The Cancer Genome Atlas (TCGA) project for colorectal cancer (CRC) is ushering in a new phase of identifying treatment strategies tailored to the molecular profile of each person's tumor. Precision medicine approaches to cancer treatment rely on the identification of molecular profiles that can be used to identify effective therapies and can be used in a targeted sequencing setting to make treatment decisions. The initial TCGA colorectal effort included 276 samples and focused on integrating data from exome sequencing with genome-wide DNA copy number alterations (CNAs), DNA methylation, and mRNA and microRNA expression. Since then a total of 626 samples have been completed with the potential to refine CRC subtypes, identify novel mutated pathways, and further functional understanding. Such a large data set presents opportunities to identify new recurrent drug targets and to stratify patients into groups that are predictive of treatment response. However, large data sets also present substantial challenges, since hand-curation becomes intractable, while computational tools can be overwhelmed by hypermutation and copy number changes. Here we present a comprehensive molecular analysis of all 626 TCGA colorectal cancer samples, including exome sequencing, CNAs, DNA methylation, and mRNA expression. For each data type, we identified recurrently altered genes. Using MutSigCV on 525 samples yielded 27 and 87 significantly mutated genes in non-hypermutated and hypermutated samples, respectively, a substantial increase over the 15 and 17 somatically recurrently mutated genes identified using MutSig in non-hypermutated and hypermutated samples, respectively, in the previously published TCGA colorectal study. For example, PTEN, a known tumor suppressor, was not reported as significantly recurrently mutated in the initial TCGA non-hypermutated set; however, it was in the larger non-hypermutated set, demonstrating the power of a larger data set for assessing the significance and relative frequency of mutations in the context of known subtypes. In addition, we integrated the somatic mutation data, copy number data, LOH data, and hyper-methylation data to identify genes, like MLH1, that are recurrently disrupted by different mechanisms. We also considered somatic mutations that are likely gain-of-function mutations based on nonrandom clustering; and we used recurrent indels to identify loss-of-function drivers in samples positive for microsatellite instability (MSI). We further classified each sample using the previously identified subtypes – BRAF+, KRAS+, APC+, CTNNB1+ (beta-catenin+), TGFBR2/SMAD4+, PTEN+ and PIK3CA+, and R-spondin fusion positive, as well as CpG Island Methylator Phenotype (CIMP) and MSI - in order to refine the relevant molecular signatures driving CRC etiology and thereby prevention and treatment paradigms. Citation Format: Catherine S. Grasso, Eve Shinbrot, Ming Yu, Max Liesersen, Mark Chaisson, Andrew Chan, Charles Connolly, James Dai, Margaret Du, Charles Fuchs, Levi Garraway, Marios Giannakis, Tabitha Harrison, Li Hsu, Jeroen Huyghe, Jasmine Mu, Shuji Ogino, Colin Pritchard, Stephen Salipante, Wei Sun, Syed H. Zaidi, Ni Zhao, William Grady, Ben Raphael, Thomas Hudson, David Wheeler, Ulrike Peters. Refining the molecular profile of colorectal tumors to expand prevention and treatment opportunities. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 136.

Abstract IA14: Targeting the Wnt signaling pathway in cancer

Abstract Mutations in the adenomatous polyposis coli (APC) gene, resulting in aberrant Wnt pathway activation, were first identified in hereditary and sporadic colorectal cancers in 1991. Today, 24 years later, there are still no approved Wnt pathway inhibitors on the market. In contrast, oncogenic BRAF mutations were discovered in various solid tumors in 2002. Within 9 years the first RAF inhibitor, vemurafenib, received FDA approval for the treatment of BRAF-mutant metastatic melanoma. Why has it been so difficult to develop Wnt pathway inhibitors? The first and most significant challenge has been to identify protein targets within the Wnt pathway that are amenable to pharmacologic inhibition. Other prominent signaling pathways dysregulated in cancer (for example, the MAPK pathway) consist of a series of activating phosphorylation events carried out by kinases, many of which can be inhibited by small molecules. However, the critical regulatory event in the Wnt pathway is the degradation of the transcriptional co-activator β-catenin by the β-catenin destruction complex, composed of APC, Axin, casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). Instead of activating the pathway, the kinases CK1 and GSK3 serve an inhibitory role, sequentially phosphorylating the amino terminus of β-catenin, triggering its ubiquitination and degradation. Confident that the Wnt pathway was important for tumorigenesis, albeit difficult to drug, Novartis initiated a high-throughput screen to identify small molecule inhibitors of Wnt secretion without a priori knowledge of their protein targets. This effort yielded WNT974, a potent and selective inhibitor of Porcupine, the O-acyltransferase that palmitoylates Wnt ligands, enabling them to be secreted. With the first synthesis of WNT974 in 2009, and efforts by Novartis and others to target additional upstream Wnt pathway components (including Frizzled, LRP6 and R-spondin), a second challenge had to be faced. Upstream Wnt pathway inhibitors are unlikely to be efficacious in tumors harboring downstream mutations in APC or β-catenin. Which, if any, human cancers are Wnt ligand-dependent, and therefore, likely to be sensitive to upstream Wnt pathway inhibitors? Recent insights into how the R-spondin/Lgr5/Rnf43 module regulates surface levels of Wnt receptors provided a critical first step towards a patient selection strategy. We now know that inactivating mutations in the Wnt antagonist RNF43 occur frequently in microsatellite-unstable colorectal, gastric, and endometrial cancers (80%, 55%, and 51%, respectively), pancreatic ductal adenocarcinoma (10%), and liver fluke-associated cholangiocarcinoma (9%). Its homolog ZNRF3 is commonly mutated in adrenocortical carcinoma (21%), and fusions that increase the expression of the Wnt agonists RSPO2 or RSPO3 are reported in 10% of colorectal cancers. Our preclinical studies in cell line and patient-derived xenograft models have confirmed that inactivating mutations of RNF43 or R-spondin fusions confer Wnt ligand dependency. Currently, a Phase 1 clinical trial with WNT974 is underway in patients with advanced cancer. Preliminary clinical data suggest WNT974 has a manageable safety profile, with dysguesia (49%) as the most common adverse event suspected to be drug related. Based on pharmacokinetic, pharmacodynamic and tolerability data, a recommended dose for expansion of 10 mg QD has been selected. In response to the emerging scientific evidence implicating the R-spondin/Lgr5/Rnf43 module in a wide variety of cancers, we adapted the clinical trial design, restricting the expansion part to patients harboring upstream Wnt pathway mutations. While there was no evidence of WNT974 anti-tumor activity in the dose escalation, one patient in the dose expansion with an RNF43-mutant appendiceal cancer had stable disease with tumor shrinkage of -27%, raising the possibility that WNT974 may have anti-tumor activity in a molecularly selected population. The intense efforts to understand the basic biology of the Wnt pathway and Wnt-driven cancers have made progress toward development of Wnt pathway inhibitors possible. Citation Format: Margaret McLaughlin. Targeting the Wnt signaling pathway in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA14.

182 TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells

Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics.

185 Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation

Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics.

181 Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt targeted drug development

Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics.

184 Molecular profiling of heterogeneous tumor cells

Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics.

Abstract 2408: Identification of R-Spondin fusions in various types of human cancer

Abstract Autocrine or paracrine constitutive Wnt pathway activation occurs at a high frequency in several tumor types. The R-spondin (RSPO) protein family is comprised of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homology. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members RSPO2 and RSPO3 occur in ∼10% of colon tumors. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 lung cancer, 81 colorectal cancer, 71 head &amp; neck, 11 esophageal, 92 ovarian cancer, and 103 breast cancer FFPET samples for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), and PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1), a fusion which is expected to produce a functional RSPO2 protein driven by the EIF3E promoter, was identified in ∼1-2% of most of cancer types with the exception of breast cancer. The PTPRK(e1)-RSPO3(e2) fusion was expressed by ∼1-11% of the samples in the different cancers, making it the most prevalent of the fusions. PTPRK(e1)-RSPO3(e2) fusion is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. The PTPRK(e7)-RSPO3(e2) fusion is also an in-frame fusion in which the RSPO3 native signal peptide is replaced by the secretion signal of PTPRK. The PTPRK(e7)-RSPO3(e2) was the least prevalent of all the fusions, positive samples were found exclusively in the head and neck (∼2%) and breast cancer samples (∼2%). All of the fusions detected were mutually exclusive. The RSPO gene fusions identified may provide new potential opportunities for therapeutic intervention. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Joseph Portale, Dana Gaffney, Christopher Moy, Suso Platero, Matthew V. Lorenzi, Jayaprakash Karkera. Identification of R-Spondin fusions in various types of human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2014-2408

RSPO fusion transcripts in colorectal cancer in Japanese population

R-spondin (RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4%) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population.

Abstract B03: Identification of R-spondin fusions in NSCLC.

Abstract Autocrine Wnt signaling has been reported in a significant fraction of NSCLC. The R-spondin (RSPO) protein family is a small family of four secreted growth factors. The four paralogs share 40–60% pairwise amino acid sequence identity and are predicted to share substantial structural homologies. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members of RSPO2 and RSPO3 that occur in ~10% of colon tumors and are mutually exclusive with other WNT pathway genetic alterations. In this study we developed a TaqMan qRT-PCR-based approach to evaluate systematically the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 NSCLC samples that included 197 squamous and 127 adenocarcinoma subtypes for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1) was identified in 1% and this fusion transcript is expected to produce a functional RSPO2 protein driven by the EIF3E promoter. The PTPRK(e1)–RSPO3(e2) transcript found in 2% and is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. Interestingly, all the fusions were detected only in the squamous subtype of NSCLC. These findings suggest an important role for dysregulated Wnt-β-catenin signaling in lung cancer and identify a new driver segment in NSCLC for therapeutic intervention. Citation Format: Jayaprakash Karkera, Gabriela Martinez, Katherine Bell, Joseph Portale, Dana Gaffney, Matthew V. Lorenzi, Suso Platero. Identification of R-spondin fusions in NSCLC. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B03.

R-spondin Fusions Activate the WNT Pathway in Colon Cancer

Abstract Gene fusions involving R-spondins occur in 10% of colon cancers.

Recurrent R-spondin fusions in colon cancer

Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.