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Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.

This file includes the Suppl. Methods, Table (1) and References

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Background: Pancreatic cancer stands as one of the deadliest tumors, in part due to the limited efficacy of currently existing therapies. Pancreatic cancer is characterized at the genomic level by the almost universal presence of KRAS mutations. Phenotypically, it features an incipient desmoplastic stroma with a protein component formed by collagens, fibronectin and laminins. A member of the laminin family, LAMININ γ2 or LAMC2, was previously identified by our group as part of a cross-tumors KRAS signature whose high expression was a marker of poor survival in PDAC patients. While a role for LAMC2 in migration and invasion has been previously described, little is known about a potential function in cell proliferation and viability. Moreover, LAMC2 inhibition has been shown to enhance the activity of conventional chemotherapeutic agents. However, whether its abrogation can increase the effect of targeted therapies is yet to be defined. Finally, the direct relationship between LAMC2 and the KRAS oncogene network is yet to be defined. Methods: A meta-analysis of several human PDAC data sets and survival analysis of the TCGA data were performed to query the expression levels and prognosis significance of LAMC2. Human and mouse PDAC cell lines as well as a mouse model of PDAC (KrasLSLG12D, Tp53f/f, Ptf1aCre) were used to assess LAMC2 expression. In vitro (2D and 3D organoids) and in vivo models derived from human and mouse PDAC cell lines or primary tumors were used to define the functional role of LAMC2 in PDAC. Cellular and molecular analysis were deployed to dissect the mechanism of action of LAMC2 in PDAC. A dual pharmacological combination based on LAMC2-regulated effectors was also tested in human and mouse pancreatic cancer models. Results: At the clinical level, we observed that LAMC2 is overexpressed in human PDAC patients with regard to normal tissue. The overexpression of LAMC2 was recapitulated in human and mouse cell lines. Genetic depletion of LAMC2 had an adverse effect in 2D proliferation, clonogenic efficiency, 3D organoid growth, and PDAC-based xenograft/allograft tumor development. This deleterious effect was driven by an induction in apoptosis and a decrease in S phase consistent across species. Mechanistically, LAMC2 was linked to the KRAS pathway via transcriptional regulation of the transcription factor FOSL1/AP1, which was also a member of the cross-tumors signature. Furthermore, LAMC2 controlled a gene signature that overlaps with KRAS- and FOSL1-regulated gene signatures, which was also highly expressed in various pancreatic cancer data sets, and includes the targetable kinase AXL. Lastly, concomitant pharmacological inhibition of AXL and the FOSL1 upstream regulator MEK1/2 displayed a more adverse effect on human and pancreatic pancreatic cancer than each single drug alone. Conclusion: These data suggest that LAMC2 is a molecular target tightly linked to KRAS oncogene signaling that regulates downstream effectors amenable to the development of combination therapies. Citation Format: Shruthi Narayanan, Oihane Erice, Iker Feliu, Caterina Vicentini, Rodrigo Entrialgo-Cadierno, Karmele Valencia, Elisabet Guruceaga, Purvesh Khatri, Vicenzo Corbo, Silvestre Vicent Cambra, Mariano Ponz-Sarvise. Identification of a LAMC2-regulated network featuring targetable effectors for dual therapies in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-089.