Billadeau, Daniel D.

Pontocerebellar hypoplasia (PCH) is a group of rare neurodevelopmental disorders with limited diagnostic and therapeutic options. Mutations in WDR11, a subunit of the FAM91A1 complex, have been found in patients with PCH-like symptoms; however, definitive evidence that the mutations are causal is still lacking. Here, we show that depletion of FAM91A1 results in developmental defects in zebrafish similar to that of TBC1D23, an established PCH gene. FAM91A1 and TBC1D23 directly interact with each other and cooperate to regulate endosome-to-Golgi trafficking of KIAA0319L, a protein known to regulate axonal growth. Crystal structure of the FAM91A1–TBC1D23 complex reveals that TBC1D23 binds to a conserved surface on FAM91A1 by assuming a Z-shaped conformation. More importantly, the interaction between FAM91A1 and TBC1D23 can be used to predict the risk of certain TBC1D23-associated mutations to PCH. Collectively, our study provides a molecular basis for the interaction between TBC1D23 and FAM91A1 and suggests that disrupted endosomal trafficking underlies multiple PCH subtypes.

Abstract The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of VPS35L, VPS26C and VPS29, together with the CCC complex comprising CCDC22, CCDC93, and COMMD proteins, plays a crucial role in this process. The precise mechanisms underlying Retriever assembly and its interaction with CCC have remained elusive. Here, we present the first high-resolution structure of Retriever determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog, Retromer. By combining AlphaFold predictions and biochemical, cellular, and proteomic analyses, we further elucidate the structural organization of the entire Retriever-CCC complex and uncover how cancer-associated mutations disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with Retriever-CCC-mediated endosomal recycling.

Abstract Metastatic pancreatic cancer patients who previously progressed on nab-paclitaxel/gemcitabine (average of 3 previous lines if treatment) were treated with elraglusib in combination with a rechallenge of this combination. The majority of patients demonstrated a prolonged progression free survival. While this early clinical evidence is highly encouraging, the biological determinants of elraglusib response are unknown. In vitro screens of elraglusib on pancreatic PDX cell lines showed that some tumor cells possess modest intrinsic resistance to elraglusib as measured by LD50 which ranged from 316.9 nM to 1080 nM in a screen of 10 cell lines. To explore this phenomena further, we induced resistance in three cell line models by growing cells in the presence of increasing concentrations of elraglusib. We then sequenced the transcriptome of the parental and resistant lines to identify potential mechanisms of elraglusib resistance. Comparison of RNA sequencing profiles identified upregulation of the Aldo-keto reductase (AKR) family of genes. This upregulation was confirmed by qRT-PCR and by immunoblot demonstrating that AKRs were upregulated at both the transcriptomic and protein level. We further quantified the AKR levels in the remainder of the 10 cell lines and found that many of the more resistant lines had high expression of AKR1C2. However, not all of the resistant lines had high expression which could be because they have a separate mechanism of resistance to elraglusib. A large body of literature has found that AKR genes provide resistance to many other small molecule inhibitors either by direct metabolism or by indirectly relieving oxidative stress. Given these results, we identify aldo-keto reductases as potentially associated with elraglusib resistance, but further investigation is warranted to establish a causal link between AKR and the resistant phenotype. Citation Format: Taylor Weiskittel, Ding Li, Lingling Han, Joseph McDermott, Benedito Caneiro, Hu Li, Daniel Billadeau, Andrew Mazar. Exploring mechanisms of resistance to elraglusib in pancreatic cancer PDX models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3857.

Abstract Elraglusib is a selective GSK-3 inhibitor that has activity on a broad array of neoplasms as well as their immune microenvironment. However the determinants of tumor response have not been elucidated. Here, we use genomic panels of cancer driver genes to predict patient response to elraglusib therapy as measured by RECIST criteria. Patient response in the Phase I 1801 trial (NCT03678883) was matched to genomic data from patient tumors, with 80 cases used to train Machine Learning models and 26 cases reserved as a test set for model evaluation. When predicting response from mutation status alone our models reached a modest accuracy of 65% in the test set, which we attributed to the sparsity of genomic data. To remedy this, we engineered pathway-based features to provide combinatorial information while ensuring feature stability. To select the optimal combination of features, we designed an iterative design process that first randomized feature combinations and then created refined models from the highest performing pathway features. This resulted in several final models with greater than 88% accuracy. We retained several models of similar accuracy to more comprehensively identify potential biomarkers and combinatorial relationships. These models were then interpreted using SHapley Additive exPlanations (SHAP) where we were able to identify highly predictive features for both the entire pan-cancer cohort and specific histologies. We found the feature constructed from the Reactome pathway “Chromatin Modifying Enzymes” frequently occurred in high performing models with a high-ranking SHAP value. This feature included genes such as SMARCA4, HDAC2, and KDM5A, and was negatively associated with patient response. Another frequently observed high-ranking pathway feature with a negative response association was based on the “Innate Immune System” Reactome pathway. Many of the features highlighted in our models were negatively associated with elraglusib response and thus could be general markers of poor prognosis. To address this, we altered our pipeline to test only positively associated features which substantially reduced our feature pool to only 285 features but still produced models with accuracies of up to 81%. Analysis of key features used by these models identified several positive markers of elraglusib including mutations in the POLE gene, which previously has been linked to DNA-damage response deficiency and anti-tumor immune response. In sum, we forecast elraglusib response for a variety of tumor histologies and simultaneously reveal potential mechanisms of elraglusib sensitivity and biological action. In future work we will investigate the utility of using POLE and other identified biomarker candidates to predict the likelihood of patient response to elraglusib, as well as whether our machine learning models will be an effective tool to guide patient stratification. Citation Format: Joseph McDermott, Taylor Weiskittel, Daniel Billadeau, Benedito Carneiro, Hu Li, Andrew Mazar. Elraglusib response prediction and mechanistic discovery using iterative machine learning. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5355.

Understanding of the evolution of metazoans from their unicellular ancestors is a fundamental question in biology. In contrast to fungi which utilize the Mon1–Ccz1 dimeric complex to activate the small GTPase RAB7A, metazoans rely on the Mon1–Ccz1–RMC1 trimeric complex. Here, we report a near-atomic resolution cryogenic-electron microscopy structure of the Drosophila Mon1–Ccz1–RMC1 complex. RMC1 acts as a scaffolding subunit and binds to both Mon1 and Ccz1 on the surface opposite to the RAB7A-binding site, with many of the RMC1-contacting residues from Mon1 and Ccz1 unique to metazoans, explaining the binding specificity. Significantly, the assembly of RMC1 with Mon1–Ccz1 is required for cellular RAB7A activation, autophagic functions and organismal development in zebrafish. Our studies offer a molecular explanation for the different degree of subunit conservation across species, and provide an excellent example of how metazoan-specific proteins take over existing functions in unicellular organisms.

Abstract Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We have shown that the actin regulatory protein HS1 is required for the stabilization of F-actin and Vav1 at the immunological synapse (IS) and for efficient Ca2+ responses. The Tec family kinase Itk also regulates actin responses, in part by facilitating the localization of Vav1 at the IS, suggesting that these proteins act in the same pathway. Using video microscopy, we found that T cells lacking Itk or HS1 behaved similarly, extending unstable actin-rich protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s SH2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, HS1 failed to localize to the IS. Since Itk is required for PLCγ1 phosphorylation and Ca2+ store release, we examined the Ca2+ signaling pathway in HS1 knockout T cells in greater detail. T cells lacking HS1 exhibited abnormal Ca2+ store release. These cells showed normal phosphorylation but altered localization of PLCγ1. We conclude that Itk-dependent recruitment of HS1 is required for actin stabilization at the IS, and directs the localization of PLCγ1. Supported by NIH F31-AI071385 to EC and NIH R01-A1065644 to JKB

Abstract IQGAP1 is a ubiquitous, cytoskeletal-interacting scaffold protein frequently overexpressed in cancer. CXCR4 is a chemokine receptor that signals upon binding stromal cell-derived factor 1 (SDF-1, CXCL12). CXCR4 regulates lymphocyte migration and development and cancer cell proliferation and metastasis. Here, we show that IQGAP1 potently enhances CXCR4’s expression, trafficking, and function in the Jurkat T cell line and epithelial cancer cells. The SDF-1-induced cell migration and activation of ERK MAPK were impaired in IQGAP1-deficient cells, even after forced CXCR4 overexpression. Exploring the mechanism, we found that SDF-1 treatment induced CXCR4 endocytosis and caused IQGAP1 to bind a-tubulin and localize to EEA-1 endosomes containing endocytosed CXCR4. In IQGAP1-deficient cells, CXCR4-containing EEA-1 endosomes accumulated at the distal ends of microtubules instead of moving towards the centrosome. This inhibited CXCR4’s signaling and cell-surface expression by preventing CXCR4 from trafficking into the Rab11 endosomes previously shown to be a critical site of CXCR4 signaling and recycling to the cell-surface. In contrast, IQGAP1 depletion had no effect on TCR trafficking, which does not require EEA-1 endosomes. Together, these results characterize a novel function of IQGAP1 as a regulator of EEA-1 endosomes, and in addition identify IQGAP1 as a critical enhancer of CXCR4 cell-surface expression and signaling in lymphocytes and epithelial cancer cells.

Abstract IQGAP1 is a scaffolding protein that integrates the actin and microtubule cytoskeletons. Although IQGAP1 has been shown to participate in numerous cellular activities in fibroblasts and epithelial cells including E-cadherin-mediated cell-cell adhesion, cell polarity, motility, proliferation, and MAP kinase signaling, its function in thymocyte development or peripheral T cell effector functions has not yet been investigated. IQGAP1 localizes to the immune synapse in CD4+ and CD8+ T cells suggesting a possible role in T cell activation. Using wild type and IQGAP1-deficient mice, we demonstrate that neither thymocyte development nor peripheral T cells numbers are affected in the absence of IQGAP1. Surprisingly, IQGAP1-deficient CD4+ T cells make significantly more IL-2 compared to wild type cells. Despite this, IQGAP1-deficient T cells show diminished T cell proliferation and increased cell death following TCR and CD28 ligation, which is most likely due to increased activation-induced cell death. Interestingly, IQGAP1-deficient T cells spread abnormally in response to anti-CD3, fail to accumulate F-actin or polarize their microtubule-organizing center (MTOC) toward anti-CD3 coated beads. Consistent with the latter observation, CD8+ T cells do show defective cell-mediated killing. Furthermore, we find that IQGAP1-deficient T cells fail to polarize following integrin stimulation. Taken together, our data identify IQGAP1 as an important mediator of proper cytoskeletal polarization during T cell activation. This work was supported by NIAID grant AI065474 to D.D.B. and NIH -T32-AI07425 pre-doctoral Immunology Training Grant to J.A. Gorman.

Abstract The NK cell activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell - target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D coated beads is impaired in the absence of CrkL. Antibody-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras-family GTPase Rap1 is required for NK cell killing and is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization and granule release.