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Supplementary figure 1. CDK6 overexpression promotes FAK inhibitor resistance in SNU668. Supplementary figure 2. CDK4/6 inhibitor enhanced the efficacy of FAK inhibitors in Cdh1-/-RHOAY42C/+ organoids. Supplementary figure 3. Combination of defactinib and palbociclib in patient-derived organoids (PDOs). Supplementary figure 4. CDK4/6 inhibitor enhanced the efficacy of FAK inhibitors in DGC cell lines. Supplementary figure 5. In vivo experiment of VS-4718 and Palbociclib combination. Supplementary figure 6. FAK inhibitor VS-4718 induces MAPK activation in DGC organoids and cell lines. Supplementary figure 7. Phospho-RTK and RAS-GTP detection in Cdh1-/-RHOAY42C/+ organoids treated with FAK inhibitors. Supplementary figure 8. MAPK inhibitor and FAK inhibitor have synergistic effect in Cdh1-/-RHOAY42C/+ organoids. Supplementary figure 9. MAPK inhibitor and FAK inhibitor have synergistic effect in PDOs. Supplementary figure 10. MAPK inhibitor and FAK inhibitor have synergistic effect in DGC cell lines. Supplementary figure 11. In vivo experiment of VS-4718 with VS-6766 or palbociclib combination.

Purpose:Diffuse gastric cancer (DGC) is an aggressive and frequently lethal subtype of gastric cancer. Because DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop targeted therapies for this gastric cancer subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC and the potential efficacy of small-molecule FAK inhibitors. However, drug resistance to monotherapy often hinders the efficacy of treatment.Experimental Design:We generated a genome-scale library of open reading frames (ORF) in the DGC model of Cdh1−/−RHOAY42C/+ organoids to identify candidate mechanisms of resistance to FAK inhibition. Compensatory activated pathways were also detected following treatment with FAK inhibitors. Candidates were investigated by cotargeting in vitro and in vivo experiments using DGC.Results:We found that cyclin-dependent kinase 6 (CDK6) promoted FAK inhibitor resistance in ORF screen. In addition, FAK inhibitor treatment in DGC models led to compensatory MAPK pathway activation. Small-molecule CDK4/6 inhibitors or MAPK inhibitors effectively enhanced FAK inhibitor efficacy in vitro and in vivo.Conclusions:Our data suggest that FAK inhibitors combined with MAPK inhibitors or CDK4/6 inhibitors warrant further testing in clinical trials for DGC.

SUMMARYGastroesophageal adenocarcinoma (GEA) is an aggressive, often lethal, malignancy that displays marked chromosomal instability (CIN). To understand adaptive responses that enable CIN, we analyzed paired normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DNA damage response (DDR), and cell cycle regulator p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, including high-level focal amplifications and whole-genome duplication (WGD). Moreover, high expression of DNA damage markerH2AXcorrelated with CIN, WGD, and inferior patient survival. By developing and implementing a composite diagnostic score that incorporatesTP53mutation status, ploidy abnormalities, andH2AXexpression, among other genomic information, we can identify GEA cell lines with enhanced sensitivity to DDR pathway inhibitors targeting Chk1/2 and Wee1. Anti-tumor properties were further augmented in combination with irinotecan (SN38) but not gemcitabine chemotherapy. These results implicate specific DDR biomarkers and ploidy abnormalities as diagnostic proxy that may predict premalignant progression and response to DDR pathway inhibitors.

Purpose: Diffuse gastric cancer (DGC) is an aggressive and frequently lethal subtype of gastric cancer. Because DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop targeted therapies for this gastric cancer subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC and the potential efficacy of small-molecule FAK inhibitors. However, drug resistance to monotherapy often hinders the efficacy of treatment. Experimental Design: We generated a genome-scale library of open reading frames (ORF) in the DGC model of Cdh1−/−RHOAY42C/+ organoids to identify candidate mechanisms of resistance to FAK inhibition. Compensatory activated pathways were also detected following treatment with FAK inhibitors. Candidates were investigated by cotargeting in vitro and in vivo experiments using DGC. Results: We found that cyclin-dependent kinase 6 (CDK6) promoted FAK inhibitor resistance in ORF screen. In addition, FAK inhibitor treatment in DGC models led to compensatory MAPK pathway activation. Small-molecule CDK4/6 inhibitors or MAPK inhibitors effectively enhanced FAK inhibitor efficacy in vitro and in vivo. Conclusions: Our data suggest that FAK inhibitors combined with MAPK inhibitors or CDK4/6 inhibitors warrant further testing in clinical trials for DGC.

Background and aimsDNA repair deficiency is a common feature of cancer. Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in solid tumors. HR deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of DNA repair pathway deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Furthermore, whether DNA repair deficient GEA have enhanced responsiveness to platinum chemotherapy and sensitivity to PARP inhibitors is not well characterized.MethodsUsing whole exome and genome sequencing data, we measured various HR deficiency-associated mutational signatures in patient specimen of gastric, esophageal and colorectal cancer specimens and gastric cancer cell lines. Gold-standard immunofluorescence assays were used to confirm HR and NER deficiency in cancer cell lines. The relationship between PARP inhibitor treatment and tumor response was evaluated in patients with gastric cancer. Drug sensitivity was determined using standard in vitro cell culture assays. Single-cell RNA-sequencing was performed to evaluate gastric cancer response to commonly used chemotherapeutics.ResultsWe found that a significant subset of GEA, but very few colorectal tumors, show evidence of HR deficiency by mutational signature analysis (HRD score). Gastric cancer cell lines with high HRD mutational signature scores demonstrated functional HR deficiency by RAD51 assay and increased sensitivity to platinum and PARP inhibitors. There was a positive association between HRD scores and tumor response in patients with gastric cancer treated with a PARP inhibitor on a clinical trial. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by DDB2 proteo-probe assay. Single-cell RNA-sequencing revealed that, in addition to inducing general apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, which may explain the outlier sensitivity.ConclusionA subset of upper gastrointestinal tumors have genomic features of HR and NER deficiency and therefore may be more likely to benefit from platinum chemotherapy and PARP inhibition.

Simple Summary Recent evidence establishes that gastric and esophageal (GE) adenocarcinomas are similar cancers at cellular, genomic, and epigenomic levels. Human GE adenocarcinomas develop TP53 mutations at early stages of malignant progression. This contrasts with other gastrointestinal adenocarcinomas, including sporadic colorectal and pancreatic adenocarcinomas, where TP53 alterations occur late. Exposure of the esophagus and stomach to environmental risk factors contributes to the selection of early TP53 mutations and subsequent chromosomal instability, which then lead to activation of mitogen and cell cycle pathways in GE adenocarcinomas by way of focal amplifications rather than mutations. While early TP53 mutations enable GE adenocarcinoma development, they also expose therapeutic vulnerabilities that should be prime for targeted therapy directed against the DNA damage response. Abstract Gastric and esophageal (GE) adenocarcinomas are the third and sixth most common causes of cancer-related mortality worldwide, accounting for greater than 1.25 million annual deaths. Despite the advancements in the multi-disciplinary treatment approaches, the prognosis for patients with GE adenocarcinomas remains poor, with a 5-year survival of 32% and 19%, respectively, mainly due to the late-stage diagnosis and aggressive nature of these cancers. Premalignant lesions characterized by atypical glandular proliferation, with neoplastic cells confined to the basement membrane, often precede malignant disease. We now appreciate that premalignant lesions also carry cancer-associated mutations, enabling disease progression in the right environmental context. A better understanding of the premalignant-to-malignant transition can help us diagnose, prevent, and treat GE adenocarcinoma. Here, we discuss the evidence suggesting that alterations in TP53 occur early in GE adenocarcinoma evolution, are selected for under environmental stressors, are responsible for shaping the genomic mechanisms for pathway dysregulation in cancer progression, and lead to potential vulnerabilities that can be exploited by a specific class of targeted therapy.

Background and Aims Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC. Methods Intestinal tissue from transgenic mice and patients were analyzed by means of histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding were determined with the use of chromatin immunoprecipitation sequencing. Results We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell–like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including PROM1. SOX9 up-regulates prominin 1 (PROM1) via a Wnt-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 uses its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop. Conclusions These studies establish SOX9 as a central regulator of an enhancer-driven stem cell–like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.

Gastric and esophageal (GE) adenocarcinomas are the third and sixth most common causes of cancer-related mortality worldwide, accounting for greater than 1.25 million annual deaths. Despite the advancements in the multi-disciplinary treatment approaches, the prognosis for patients with GE adenocarcinomas remains poor, with a 5-year survival of 32% and 19%, respectively, mainly due to the late-stage diagnosis and aggressive nature of these cancers. Premalignant lesions characterized by atypical glandular proliferation, with neoplastic cells confined to the basement membrane, often precede malignant disease. We now appreciate that premalignant lesions also carry cancer-associated mutations, enabling disease progression in the right environmental context. A better understanding of the premalignant-to-malignant transition can help us diagnose, prevent, and treat GE adenocarcinoma. Here, we discuss the evidence suggesting that alterations in TP53 occur early in GE adenocarcinoma evolution, are selected for under environmental stressors, are responsible for shaping the genomic mechanisms for pathway dysregulation in cancer progression, and lead to potential vulnerabilities that can be exploited by a specific class of targeted therapy.

The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration.

β1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes like cancer cell invasion. Here we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with β1-integrin and its role in recycling of the active but not inactive β1-integrin receptors. Silencing of GGA2 limits active β1-integrin levels in focal adhesions and decreases cancer cell migration and invasion congruent with its ability to regulate the dynamics of active integrins. Using the proximity-dependent biotin identification (BioID) method, we identify two RAB family small GTPases, RAB13 and RAB10, associating with GGA2 and β1-integrin. Functionally, RAB13 silencing triggers the intracellular accumulation of active β1-integrin, reduces integrin activity, in focal adhesions, and cell migration, similarly to GGA2 depletion, indicating that both facilitate active β1-integrin recycling the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.

Human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. Endosomal trafficking of many other receptor tyrosine kinases regulates their oncogenic signaling, but the prevailing view is that HER2 is retained on the cell surface. Here we reveal that in cancer cells Sortilin related receptor 1 (SORLA; SORL1) forms a complex with HER2 and regulates its subcellular distribution by promoting recycling of endosomal HER2 back to plasma membrane. Expression of SORLA in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA targets HER2 to late endosomal/lysosomal compartments, impairs HER2-driven signaling and in vivo tumor growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

The transmembrane protease ADAM9 is frequently upregulated in human cancers, and it promotes tumour progression in mice. In vitro, ADAM9 regulates cancer cell adhesion and migration by interacting with integrins. However, how ADAM9 modulates integrin functions is not known. We here show that ADAM9 knockdown increases β1 integrin levels through mechanisms that are independent of its protease activity. In ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with β1 integrin, and both internalization and subsequent degradation of β1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on β1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction in cell adhesion and migration in these cells. Taken together, our data provide mechanistic insight into the ADAM9-integrin interaction, demonstrating that ADAM9 regulates β1 integrin endocytosis. Moreover, our findings indicate that the reduced migration of ADAM9-silenced cells is, at least in part, caused by the accumulation and altered activity of β1 integrin at the cell surface.

Integrins are a family of transmembrane cell surface molecules that constitute the principal adhesion receptors for the extracellular matrix (ECM) and are indispensable for the existence of multicellular organisms. In vertebrates, 24 different integrin heterodimers exist with differing substrate specificity and tissue expression. Integrin–extracellular-ligand interaction provides a physical anchor for the cell and triggers a vast array of intracellular signalling events that determine cell fate. Dynamic remodelling of adhesions, through rapid endocytic and exocytic trafficking of integrin receptors, is an important mechanism employed by cells to regulate integrin–ECM interactions, and thus cellular signalling, during processes such as cell migration, invasion and cytokinesis. The initial concept of integrin traffic as a means to translocate adhesion receptors within the cell has now been expanded with the growing appreciation that traffic is intimately linked to the cell signalling apparatus. Furthermore, endosomal pathways are emerging as crucial regulators of integrin stability and expression in cells. Thus, integrin traffic is relevant in a number of pathological conditions, especially in cancer. Nearly a decade ago we wrote a Commentary in Journal of Cell Science entitled ‘Integrin traffic’. With the advances in the field, we felt it would be appropriate to provide the growing number of researchers interested in integrin traffic with an update.

The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration.

Summary Inappropriate MET tyrosine kinase receptor signaling is detected in almost all types of human cancer and contributes to malignant growth and MET dependency via proliferative and antiapoptotic activities. Independently, Tensin-4 (TNS4) is emerging as a putative oncogene in many cancer types, but the mechanisms of TNS4 oncogenic activity are not well established. Here, we demonstrate that TNS4 directly interacts with phosphorylated MET via the TNS4 SH2-domain to positively regulate cell survival, proliferation, and migration, through increased MET protein stability. In addition, TNS4 interaction with β1-integrin cytoplasmic tail positively regulates β1-integrin stability. Loss of TNS4 or disruption of MET-TNS4 interaction triggers MET trafficking toward the lysosomal compartment that is associated with excessive degradation of MET and triggers MET-addicted carcinoma cell death in vitro and in vivo. Significant correlation between MET and TNS4 expression in human colon carcinoma and ovarian carcinoma suggests TNS4 plays a critical role in MET stability in cancer., Graphical Abstract, Highlights • A direct interaction is identified between MET and Tensin-4 • TNS4 protects MET from degradation, thus promoting its oncogenic activity • TNS4 and MET are significantly coexpressed in human carcinomas • Loss of TNS4 inhibits survival of MET-dependent tumors, Muharram et al. demonstrate that a direct interaction between MET and TNS4, a recently characterized oncogene, inhibits MET trafficking and induces MET stability, cell proliferation, migration, and survival of MET-addicted carcinomas in vivo. A significant correlation among MET-TNS4 levels in human carcinomas suggests clinical relevance of TNS4-dependent MET stabilization.

Background: The emergence of influenza A/H1N1/2009 is alarming. The severity of previous epidemics suggests that the susceptibility of the human population to H1N1 is directly proportional to the degree of changes in hemagglutinin/HA and neuraminidase/NA; therefore, H1N1/2009 and H1N1/2008 were analyzed for their sequence as well as structural divergence. Methodology: The structural and sequence divergence of H1N1/2009 and H1N1/2008 strains were analyzed by aligning HA and NA amino acid sequences by using ClustalW and ESyPred3D software. To determine the variations in sites of viral attachment to host cells, a comparison between amino acid sequences of HA and NA glycosylation sites was performed with NetNGlyc software. The antigenic divergence was executed by CTL epitope prediction method. Results: The amino acid homology levels of H1N1/2009 were 20.32% and 18.73% compared to H1N1/2008 for HA and NA genes, respectively. In spite of the high variation in HA and NA amino acid composition, there was no significant difference in their structures. Antigenic analysis proposes that great antigenic differences exist between both the viral strains, but no addition of a new site of glycosylation was observed. Conclusions: To our knowledge, this is the first report suggesting that the circulating novel influenza virus A/H1N1/2009 attaches to the same glycosylation receptor sites as its predecessor influenza A/H1N1/2008 virus, but is antigenically different and may have the potential for initiating a significant pandemic. Our study may facilitate the development of better therapeutics and preventive strategies, as well as impart clues for novel H1N1 diagnostic and vaccine development.