Biology subjects

Gutkind, J. S.

Publications and source records attributed to Gutkind, J. S..

At least 19 recordsLinked to original sources

bioRxiv2026

A reversible GNAS-PKA-RUNX2 checkpoint governs skeletal stem cell fate in fibrous dysplasia

Fibrous dysplasia (FD) arises from activating GNAS mutations that increase cAMP signaling in skeletal stem/progenitor cells (SSPCs), but the underlying mechanisms and whether the resulting fate imbalance can be reversed remain unclear. Using controlled GNAS induction with transcriptomic, pharmacologic, and cell-fate analyses, we identify a PKA-dependent checkpoint governing SSPC lineage commitment. GNASR201C redirects SSPCs toward osteoclastogenesis while suppressing osteoblast maturation; genetic or pharmacologic PKA inhibition reverses both effects, revealing this fate decision is dynamically reversible. Mechanistically, PKA phosphorylates RUNX2, the master regulator of osteogenesis, enforcing its cytoplasmic retention and blocking osteogenic transcription; a non-phosphorylatable RUNX2 mutant restores nuclear localization and osteoblast differentiation, defining RUNX2 phosphorylation as a disease-initiating molecular switch. Critically, PKA inhibition in vivo reduces fibrosis and osteoclast burden in established bone lesions, demonstrating reversibility even in advanced FD. These findings define a druggable GNAS-PKA-RUNX2 checkpoint and identify PKA inhibition as a strategy to reverse existing disease in FD.

cell biology↗

bioRxiv2026

Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease

cAMP-dependent Protein Kinase A (PKA) is a master regulator of cell signaling involved in energy metabolism, synaptic plasticity, and stress response. Dysregulated PKA signaling is implicated in diseases including neurodegeneration and cancer. PKA catalytic activity is regulated by two nonredundant regulatory subunits, Type I (RI/RI{beta}) and Type II (RII/RII{beta}), whose divergent functions are not fully understood. We generated double-knockout (KO) cell lines of RI/RI{beta} and RII/RII{beta} subunits and performed multiplexed MS-based proteomic and phosphoproteomic profiling under basal and glucose-perturbed conditions. We found that RI and RII loss drives distinct, and often opposite, remodeling of the cellular proteome and phosphoproteome. While both mutants blunted metabolic flexibility to glycolytic stressors and stimuli, RI and RII KO cells exhibited elevated and depressed glycolytic signaling, respectively. Interestingly, RI KO increased the abundance and kinase activity of the PKA catalytic subunit C isoform, leading to an increase in PKA substrate phosphorylation, whereas RII KO decreased the abundance, kinase activity, and substrate phosphorylation by the catalytic subunit C{beta} isoform. Notably, one of the most differentially affected PKA sites between RI and RII KOs maps to Tau, whose hyperphosphorylation is a hallmark of Alzheimers disease. Loss of RI increased Tau phosphorylation, which was not only caused by increased PKA catalytic activity, but also a higher binding affinity of Tau to RII subunits on the negatively-charged flexible linker region. Overall, the present study demonstrates that PKA RI and RII subunits play nonredundant roles in modulating PKA activity, metabolic flexibility, and phospho-regulation of key disease-associated substrates such as Tau.

biochemistry↗

bioRxiv2026

mTOR inhibition augments antitumor immune effector response by reprogramming the TP53-mutant, immune-cold HNSCC tumor microenvironment.

IntroductionResistance to immunotherapy remains a major clinical challenge in TP53-mutant head and neck squamous cell carcinoma (HNSCC), a disease subset characterized by immune exclusion, high recurrence, and poor outcomes. Given the constitutive activation of PI3K/AKT/mTOR signaling in TP53-mutant HNSCC and its role in disease progression, we investigated whether the mTOR inhibition (mTORi) could overcome immune resistance and improve outcomes. MethodsWe evaluated the effects of mTOR inhibitor everolimus in TP53-mutant, anti-PD-1-resistant syngeneic HNSCC cell lines and syngeneic tumor models. Tumor microenvironment (TME) changes, including immune cell infiltration, immune checkpoint expression, and key pathways associated with immune suppression and angiogenesis were assessed to define the mechanisms underlying TME remodeling. ResultsEverolimus significantly suppressed tumor growth in syngeneic HNSCC models. At the cellular level, everolimus significantly increased intratumoral CD8+ T cell and dendritic cell (DC) infiltration while reducing the accumulation of regulatory T cells (Tregs). Mechanistically, everolimus induced a cytokine/chemokine response, marked by increased TNF-/CXCL10 expression, leading to enhanced immune infiltration. Everolimus also inhibited the HIF-1/VEGFA angiogenic axis, a central driver of immune exclusion and myeloid-derived suppressor cell (MDSC) recruitment. Furthermore, everolimus treatment attenuated PD-1/PD-L1 signaling by reducing PD-1 and PD-L1 expression, respectively, in T cells and tumor cells, thereby restoring T-cell cytotoxic competence. DiscussionThese findings demonstrate that mTORi with everolimus reverses multiple mechanisms of immune resistance in TP53-mutant HNSCC by promoting immune cell recruitment, suppressing immunosuppressive pathways, and enhancing anti-tumor T cell activity. Collectively, these results support mTORi as a mechanistically rational strategy for reprogramming immune resistance in TP53-mutant HNSCC and provide a strong preclinical rationale for combining everolimus with immune therapy in patients who are likely to fail immunotherapy.

cancer biology↗

bioRxiv2026

Pancreatic Gαs ablation disrupts tissue architecture and YAP signaling and unveils a compensatory regenerative response

Diabetes mellitus is characterized by chronic hyperglycemia and loss of pancreatic {beta}-cell function and mass. Current therapies focus on {beta}-cell protection and regeneration, led by GLP-1 receptor agonists. The G protein -subunit (Gs) acts as a key signaling node downstream of numerous GPCRs, integrating diverse signals that impact {beta}-cell mass and function. Elucidating the integrative role of pancreatic Gs signaling is thus crucial for understanding {beta}-cell biology. Our map of the pancreatic Gs-coupled GPCR landscape reveals sophisticated, cell-type-specific networks, positioning Gs as a central hub for intra-pancreatic communication. Previous studies in mice with {beta}-cell-specific or whole-pancreatic Gs deletion demonstrated reduced {beta}-cell mass, impaired insulin secretion, and glucose intolerance. The stronger phenotype in the whole-pancreas model--marked by -cell expansion and abnormal distribution--points to a crucial role for Gs in differential control of postnatal - and {beta}-cell proliferation. Here, we analyze the organ-wide consequences of Gs deletion using pancreas-specific Gs knockout mice (PGsKO). Consistent with prior findings, PGsKO mice exhibit reduced weight gain from four weeks and severe diabetes due to decreased {beta}-cell mass and concomitant -cell expansion. Furthermore, Gs loss induces profound architectural and functional defects in the exocrine pancreas, linked to YAP reactivation in acinar cells. Importantly, we observed attempted {beta}-cell regeneration in PGsKO mice. Although insufficient to reverse diabetes, our results delineate the full pancreatic phenotype that may facilitate these regenerative efforts and suggest that strategically biasing GPCR signaling network away from Gs could be a viable strategy to promote {beta}-cell regeneration from other pancreatic cell types. ARTICLE HIGHLIGHTSO_LIGs is a central signaling hub that integrates diverse GPCR inputs across pancreatic cell types, yet its organ-wide role remained poorly defined. C_LIO_LIWe addressed how pancreas-wide Gs deletion disrupts both endocrine and exocrine compartments, and whether regenerative programs are engaged. C_LIO_LIGs loss caused severe diabetes through {beta}-cell loss and -cell expansion, induced profound exocrine defects with YAP reactivation, and triggered attempted {beta}-cell regeneration from ducts and potentially other cell types. C_LIO_LIOur findings suggest that strategically biasing GPCR signaling away from Gs could promote regeneration from non-{beta}-cell sources, offering new therapeutic avenues for diabetes. C_LI

cell biology↗

bioRxiv2026

The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation.

The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA) to induce type I interferon (IFN) response, which is essential for mounting the antiviral response. However, STING activation during viral infection is often insufficient to achieve complete viral clearance, suggesting the existence of additional mechanisms that evade its activity. Here, we identified COX2/PGE2 as a negative regulator of STING activation, particularly in response to arising cytosolic mitochondrial DNA (mtDNA) generated during HSV-1 infection. Mechanistically, PGE2, through the EP4-cAMP-PKA axis, induces mitophagy to remove defective mitochondria and hence prevent the accumulation of immunostimulatory cytosolic mtDNA, thereby dampening STING-mediated type I IFN and antiviral response. Furthermore, we identified STOML2 as a downstream target of PKA that connects mitochondrial quality control with the regulation of innate immune signaling. Together, our findings establish the COX2/PGE2/PKA axis as a negative regulator of mtDNA-STING signaling that may be targeted to potentiate STING-mediated type I IFN and innate immunity. O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/716411v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c39d56org.highwire.dtl.DTLVardef@18e7c0eorg.highwire.dtl.DTLVardef@172b353org.highwire.dtl.DTLVardef@15b215a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

cell biology↗

bioRxiv2026

Computed atlas of the human GPCR-G protein signaling complexes

Experimental mapping of G protein-coupled receptors (GPCR)-G protein signaling coupling has illuminated hundreds of receptors, yet the coupling specificity of a large fraction of this large receptor family remains unknown, thereby preventing the development of new GPCR-targeting therapies. Here, we used AlphaFold3 (AF3) to predict the 3D structures of the human GPCRome in complex with heterotrimeric G proteins. We used experimental GPCR-G protein binding data to show that AF3 predictions significantly discriminate between positive and negative binders, and used 3D structural features to train a machine learning (ML) algorithm to predict coupling potency. Interpretation of the ML model helped discriminate universal features governing the strength of G protein coupling from those determining binding specificity. We computationally illuminated the coupling preferences of 180 non-olfactory GPCRs (non-OR) with previously unreported transduction mechanisms and experimentally validate the predicted couplings for multiple previously uncharacterized GPCRs, including QRFPR, GPR50, GPR37, GPR37L1 and GPRC5A. Our predictions established that Gi/o is the most prevalent coupling among non-OR GPCRs, which is often co-occurring with Gq/11 and, to a lesser extent, G12/13 signaling. Gs coupling is less common and restricted to specific clusters within the non-OR GPCRome phylogeny, likely due to stricter structural requirements for its binding. We also computed G protein complexes for over 400 ORs, establishing Gs as the most prevalent coupling. ORs are predicted to bind to Gs with a simpler interface compared to non-ORs, ultimately leading to energetically less stable complexes. Additionally, we predict recurrent bindings to Gq/11 and Gi/o proteins for ORs, suggesting potentially novel ORs signaling mechanisms. We exploited the GPCRome coupling atlas to interpret healthy and cancer expression data, revealing the coupling of most GPCR-G protein co-expressed pairs. This analysis highlights a richer coupling repertoire in healthy tissues compared to cancer, likely reflecting the high signaling requirements of specialized normal cell functions, which are lost in most cancer cells due to their de-differentiated state or under cancer selection processes. In summary, this study provides the first computational 3D atlas of the human GPCR-G protein transductome, thereby illuminating the signaling mechanisms of neglected GPCR classes and providing the basis for interpreting omics datasets from a myriad of pathological conditions, thus enabling the development of novel precision therapeutics.

bioinformatics↗

bioRxiv2026

Orthogonally targeted tumor radiosensitization using cell penetrating peptide-ATM inhibitor conjugates to stimulate anti-tumor immune responses

Tumor resistance to radiotherapy continues to be a significant problem in improving cancer patient outcomes. To overcome radioresistance, drugs that sensitize cancer cells to ionizing radiation have been tested. In theory, radiosensitizers should increase irradiated tumor kill and improve patient outcomes. In practice, the clinical utility of such drugs is curtailed by radiosensitization of peri-tumoral normal tissues causing toxicities. To address these issues, we developed an activatable cell penetrating peptide-drug conjugate to deliver a small molecule radiosensitizer with spatial precision to tumors. The activatable cell penetrating peptide (ACPP) scaffold cloaks a cell penetrating peptide-drug conjugate until it is unmasked within tumors through matrix metalloproteinase cleavage. Using antibody-drug conjugate linker chemistry, we attached the potent ataxia-telangiectasia mutated (ATM) kinase inhibitor AZD0156 to ACPP and created ACPP-AZD0156. In immune-competent murine cancer models, tumor-targeted ACPP-AZD0156 in combination with ionizing radiation stimulated tumor immune infiltration by CD8+ T cells and increased tumor control when compared to non-targeted ATM inhibitor. Mechanistically, ACPP-AZD0156 radiosensitized tumor control was dependent on the adaptive arm of the immune system. Finally, the combination of radiotherapy and ACPP-AZD0156 potentiated immune checkpoint inhibitors that resulted in durable tumor control. The therapeutic synergies of ACPP targeted ATM inhibitor to radiosensitize and stimulate anti-tumor immune responses provides a rationale for developing tumor-targeted radiosensitizer drug conjugates that restrict radiosensitization to cancer cells that then engages anti-tumor immune responses to improve cancer patient outcomes.

pharmacology and toxicology↗

bioRxiv2025

Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

Accessibility of immune cells to the tumor microenvironment (TME) in many solid tumors can be influenced by extracellular matrix (ECM) deposition, organization, and remodeling within tumor stroma. Specifically, lysyl hydroxylase-2 (LH2)-catalyzed lysine hydroxylation in type-I collagen telopeptides leads to formation of intermolecular collagen cross-links creating a stiffened and proteolytically resistant, stable ECM. Advanced head and neck squamous cell carcinomas (HNSCC) frequently exhibit desmoplasia with elevated LH2 expression, and Immune-checkpoint (ICI) therapy is effective in only <20% patients, suggesting that ECM remodeling mechanistically governs the composition and function of TME infiltrates. We show that elevated LH2 and collagen alignment promotes stromal accumulation of CD8 T-cells, and poor response to ICI in HPV-HNSCCs. Integration of clinical biopsies, transcriptomic datasets, and an immunocompetent syngeneic mouse model revealed that aligned collagen spatially restricts adhesion G protein-coupled receptor positive (ADGRG1) CD8 T-cells to activate a non-canonical GPCR-mediated mechanosensory program that drives dysfunction and exhaustion. Statement of significanceDesmoplasia is common in solid tumors, and immune checkpoint inhibitors benefit only some patients. Current biomarkers like PD-L1 and TMB have limited value. Our findings reveal a previously unrecognized collagen-ADGRG1 mechanosensory immune checkpoint, offering a clinically tractable, spatially resolved biomarker to better stratify patients for immunotherapy.

cancer biology↗

bioRxiv2025

Aberrant Hippo-YAP/TEAD signaling drives malignant transcriptional reprogramming in external auditory canal squamous cell carcinoma

PurposeExternal auditory canal squamous cell carcinoma (EACSCC) is an extremely rare malignancy. The molecular characteristics and evidence-based therapeutic strategies of EACSCC still remain to be elucidated. Experimental DesignComprehensive analyses of RNA sequencing (RNA-seq) and ChIP sequencing (ChIP-seq) utilizing YAP and H3K27Ac antibodies were performed in primary EACSCC and noncancerous ear skin samples. Functional experiments were performed in EACSCC-derived cells and Head and Neck Squamous Cell Carcinoma (HNSCC) cells in vitro and in vivo. Immunohistochemical staining of primary EACSCC tissues as well as survival analysis were conducted. ResultsRNA-seq indicated hyperactivation of YAP/TEAD-mediated transcriptional programs in EACSCC. H3K27Ac ChIP-seq suggested gained accessibility for transcription factor (TF) binding sites for TEAD, AP-1 and PITX TFs in EACSCC, and presence of EACSCC-specific super enhancers (SEs). YAP-bound SEs were involved in oncogenic transcription, including EGFR signaling. Small molecule TEAD inhibitor (smTEADi) VT104 showed significant suppression of proliferation and clonogenicity in EACSCC cells. Importantly, smTEADi not only inhibited YAP-TEAD interaction but also induced YAP-PITX2 binding, suggesting that PITX2 could represent an alternative partner TF of YAP under TEAD-inhibited conditions. Knockdown of PITX2 inhibited cell growth and migration of EACSCC and HNSCC cells, whereas overexpression of PITX2 induced expression of cell cycle, stemness, and EMT genes, as well as YAP/TAZ-TEAD target genes, and promoted tumor growth in vivo. Nuclear YAP and PITX2 expression were significantly correlated with poor prognosis of EACSCC patients. ConclusionsThis study highlighted the hyperactivation of the YAP-TEAD/PITX2 transcriptional program and its potential as a therapeutic target in EACSCC. Translational RelevanceExternal auditory canal squamous cell carcinoma (EACSCC) is an extremely rare malignancy related to chronic tissue damage and inflammation. Due to its rarity, the molecular characteristics of EACSCC are poorly understood, and evidence-based therapeutic strategies are not fully developed. Here, we provide evidence of hyperactivation of YAP/TEAD-driven transcriptional programs in EACSCC, utilizing comprehensive analyses of RNA-seq and YAP/H3K27Ac ChIP-seq in clinical tissue samples, as well as in vitro and in vivo experiments. In addition, our data suggest that the PITX2 transcription factor (TF) could represent an alternative partner TF of YAP under TEAD-inhibited conditions, which may rescue oncogenic transcription of TEAD. Importantly, YAP and PITX2 are co-expressed in EACSCC and predict poor prognosis of EACSCC patients. Our results provide a rationale for YAP-hyperactivation in EACSCC and contribute to a better understanding of this malignancy and the development of new therapeutic strategies.

cancer biology↗

bioRxiv2025

Antigenic cancer persister cells survive direct T cell attack

Drug-tolerant persister cancer cells were first reported fifteen years ago as a quiescent, reversible cell state which tolerates unattenuated cytotoxic drug stress. It remains unknown whether a similar phenomenon contributes to immune evasion. Here we report a persister state which survives weeks of direct cytotoxic T lymphocyte (CTL) attack. In contrast to previously known immune evasion mechanisms that avoid immune attack, antigenic persister cells robustly activate CTLs which deliver Granzyme B, secrete IFN{gamma}, and induce tryptophan starvation resulting in apoptosis initiation. Instead of dying, persister cells paradoxically leverage apoptotic caspase activity to avoid inflammatory death. Furthermore, persister cells acquire mutations and epigenetic changes which enable outgrowth of CTL-resistant cells. Persister cell features are enriched in inflamed tumors which regressed during immunotherapy in vivo and in surgically resected human melanoma tissue under immune stress ex vivo. These findings reveal a persister cell state which is a barrier to immune-mediated tumor clearance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/643359v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ca8905org.highwire.dtl.DTLVardef@1e9b584org.highwire.dtl.DTLVardef@8bf43eorg.highwire.dtl.DTLVardef@504918_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Data driven refinement of gene expression signatures for enrichment analysis

Gene set enrichment methods measure biological process or pathway activation in gene expression data by testing coordinate up- or down-regulation of pathway members in a ranked list of genes. These methods rely on curated, annotated gene sets whose members coordinate expression is an indicator of a process or state. We therefore developed the Molecular Signatures Database (MSigDB), a collection of expertly annotated gene sets. While using, enhancing, and expanding MSigDB, we have observed that some gene sets can lack coordinate expression, especially those derived from canonical pathways. To address this challenge, we developed gene set refinement (GSR), a data-driven approach leveraging large-scale multi-omics compendia to extract context-specific sets, deconvolve heterogeneity, and reveal multiple downstream signaling. We applied this method to address cancer biology questions, and demonstrated successful, targeted refinement of existing MSigDB gene sets.

bioinformatics↗

bioRxiv2024

Tumor-induced endothelial RhoA activation mediates tumor cell transendothelial migration and metastasis

Abstract/SummaryThe endothelial barrier plays an active role in transendothelial tumor cell migration during metastasis, however, the endothelial regulatory elements of this step remain obscure. Here we show that endothelial RhoA activation is a determining factor during this process. Breast tumor cell-induced endothelial RhoA activation is the combined outcome of paracrine IL-8-dependent and cell-to-cell contact {beta}1 integrin-mediated mechanisms, with elements of this pathway correlating with clinical data. Endothelial-specific RhoA blockade or in vivo deficiency inhibited the transendothelial migration and metastatic potential of human breast tumor and three murine syngeneic tumor cell lines, similar to the pharmacological blockade of the downstream RhoA pathway. These findings highlight endothelial RhoA as a potent, universal target in the tumor microenvironment for anti-metastatic treatment of solid tumors.

physiology↗

bioRxiv2023

Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and the heterotrimeric G{beta}{gamma} subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1{middle dot}IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

biochemistry↗

bioRxiv2023

A kinome-wide synthetic lethal CRISPR/Cas9 screen reveals that mTOR inhibition prevents adaptive resistance to CDK4/CDK6 blockade in HNSCC

The comprehensive genomic analysis of the head and neck cancer (HNSCC) oncogenome revealed frequent loss of p16INK4A (CDKN2A) in most HPV negative HNSCC lesions, often concomitant with amplification of the cyclin D1 (CCND1) gene locus. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors as single agents have shown modest effect in the clinic, even when combined with cetuximab. The aberrant activation of PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials targeting mTOR showed promising results in terms of objective responses and progression free survival. However, the clinical efficacy of mTOR inhibitors (mTORi) for advanced HNSCC patients may be limited due to intrinsic or acquired resistance. By a kinome-wide CRISPR/Cas9 screen, we identified cell cycle inhibition as a synthetic lethal target for mTORi. Combination of mTORi and palbociclib, a CDK4/6 specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor in HNSCC. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that concomitant mTOR blockade reverts the adaptive resistance to palbociclib, thereby providing a novel multimodal therapeutic option for HNSCC patients by co-targeting mTOR and CDK4/6. Our findings may have broad implications to halt the emergence of palbociclib resistance.

cancer biology↗

bioRxiv2023

Direct reprogramming of oral epithelial progenitor cells to cancer stem cells at single cell resolution in vivo

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells (TIC) at single cell resolution. TIC displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. YAP-mediated TIC programs included the activation of oncogenic transcriptional networks and mTOR signaling, and the recruitment of myeloid cells to the invasive front contributing to tumor infiltration. TIC transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

cancer biology↗

bioRxiv2023

The landscape of cancer rewired GPCR signaling axes

We explored the dysregulation of GPCR ligand signaling systems in cancer transcriptomics datasets to uncover new therapeutics opportunities in oncology. We derived an interaction network of receptors with ligands and their biosynthetic enzymes, which revealed that multiple GPCRs are differentially regulated together with their upstream partners across cancer subtypes. We showed that biosynthetic pathway enrichment from enzyme expression recapitulated pathway activity signatures from metabolomics datasets, providing valuable surrogate information for GPCRs responding to organic ligands. We found that several GPCRs signaling components were significantly associated with patient survival in a cancer type-specific fashion. The expression of both receptor-ligand (or enzymes) partners improved patient stratification, suggesting a synergistic role for the activation of GPCR networks in modulating cancer phenotypes. Remarkably, we identified many such axes across several cancer molecular subtypes, including many pairs involving receptor- biosynthetic enzymes for neurotransmitters. We found that GPCRs from these actionable axes, including e.g., muscarinic, adenosine, 5-hydroxytryptamine and chemokine receptors, are the targets of multiple drugs displaying anti-growth effects in large-scale, cancer cell drug screens. We have made the results generated in this study freely available through a webapp (gpcrcanceraxes.bioinfolab.sns.it). SignificanceComprehensive analysis of GPCR extracellular network in cancer transcriptomics datasets reveals signaling axes associated to patient survival, whose targeting is associated with growth inhibition in cancer cell lines drug sensitivity assays.

bioinformatics↗

bioRxiv2023

Tipifarnib potentiates the antitumor effects of PI3Kα inhibition in PIK3CA- and HRAS-dysregulated HNSCC via convergent inhibition of mTOR activity

Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival ranging from 6 to 18 months. For those who progress on standard of care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase inhibitor, and alpelisib, a PI3K inhibitor, in multiple molecularly defined subsets of HNSCC. We find that tipifarnib synergizes with alpelisib at the level of mTOR in PI3K-or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. Based on these findings, we have launched the KURRENT-HN trial to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. SignificanceBacked by strong mechanistic rationale, the combination of alpelisib and tipifarnib has the potential to benefit >45% of R/M HNSCC patients. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, thereby enhancing their clinical utility.

cancer biology↗

bioRxiv2022

A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors

BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF/MEK/EGFR co-targeting, we used a high throughput kinase activity mapping platform. We found that SRC kinases are systematically activated in BRAFV600E CRC following targeted inhibition of BRAF {+/-} EGFR, and that coordinated targeting of SRC with BRAF {+/-} EGFR increases efficacy in vitro and in vivo. SRC drives resistance to BRAF {+/-} anti-EGFR therapy independently of ERK signaling by inducing transcriptional reprogramming via beta-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2-loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF+EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft (PDX) models. COX2 inhibition represents a novel drug-repurposing strategy to overcome therapeutic resistance in BRAFV600E CRC.

cancer biology↗