García, K. Christopher

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality, as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncovered that the EGFR pathway component RasGRP1 acts as a CRC tumor suppressor in the context of aberrant Wnt signaling. We found that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impact biology, we next focused on CRC patients. Mining 5 different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Last, deletion of 1 or 2 Rasgrp1 alleles made CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB 80203 clinical trial showed that addition of anti-EGFR therapy to chemotherapy significantly improved outcome for CRC patients when tumors expressed low levels of RasGRP1 suppressor. In sum, our data support RasGRP1 as a biomarker in the EGFR pathway that has potential relevance to anti-EGFR therapy for CRC patients.

The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide–major histocompatibility complex (pMHC) antigen H-2K b –dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR β chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC α helices suggests a generalized orientation that is mediated by the V α domain of the TCR and rationalizes how TCRs can effectively “scan” different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2K b -dEV8, H-2K bm3 -dEV8, H-2K b -SIYR, and H-2L d -p2Ca spans a range of affinities from K d = 10 −4 to 10 −6 M for the syngeneic (H-2K b ) and allogeneic (H-2K bm3 , H-2L d ) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2K b -dEV8, H-2K bm3 -dEV8 and H-2K b -SIYR have been grown.