Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications.
Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute’s Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12–24 hr to avoid confounding effects of these preanalytical factors on IHC.
Background: Medulloblastoma is the most common malignant brain tumor of childhood and is considered a tumor with low mutational burden (1 Mut/Mb). Therefore, though the medulloblastoma genomes have been extensively characterized in literature, reports on potential hypermutations and underlying mutagenic processes in medulloblastomas are limited.Aim: In this report, we studied the landscape of mutational burden in primary and recurrent medulloblastoma. Furthermore, we wanted to understand the differences in underlying mutagenic mechanisms in medulloblastoma with low and high mutational burdens.Methods: Fifty-three primary and recurrent medulloblastoma genomic sequence were downloaded from the European Genome Archive as BAM files. Thirty-three cases were obtained from formalin-fixed paraffin-embedded tissues from pathology diagnostic archives of Spectrum Health and Cooperative Human Tissue Network. Somatic mutations were called using Mutect2, following best practices guidelines for Genome Analysis Toolkit V4. Mutational signatures were analyzed using deconstructSigs.Results: We identified nine medulloblastoma cases with high mutational burden (>5 Mut/Mb). Of them, five cases met the criteria of hypermutation (>10Mut/Mb), two of the five tumors had canonical mutations in the POLE proof-reading domain, where a large proportion of mutations in these tumor genomes contributed to signature 10.The hypermutated cases also demonstrated mutational signatures 14, 15, and 21, indicating the role of mis match repair deficiency in their mutagenesis. Of the four known molecular subgroups in medulloblastoma-SHH, WNT, Group 3, and Group 4-both the POLE-mutated cases belonged to the SHH subgroup. This report identifies rare cases of hypermutation in medulloblastoma driven by defects in DNA repair mechanisms.Conclusion: Hypermutation in medulloblastoma can impact therapeutic decisions, especially at recurrence in otherwise fatal high risk SHH-medulloblastomas. A defect
Medulloblastoma (MB) is the most common malignant brain tumor of childhood and is reported to have a low mutational burden. However, in this study, we identified nine MBs with high mutational burden by next generation sequencing. Of them, two had canonical mutations in the POLE proof-reading domain, where a large proportion of mutations in these tumor genomes contributed to signature 10. We report very rare incidences of hypermutation in MB and mechanisms driving mutagenesis. Strikingly, of the four known molecular subgroups in MB—-SHH, WNT, Group 3, and Group 4—both the POLE-mutated MBs belonged to the SHH subgroup.
The somatic KRAS mutations found in ~25% of human cancers commonly encode amino acid substitutions at codons 12, 13, and 61. Each of these mutations lead to elevated levels of active Ras-GTP by reducing intrinsic Ras GTPase activity and conferring resistance to GTPase activating proteins (GAPs). Amino acids 12, 13, and 61 play key roles in binding the γ phosphate of GTP and facilitating GAP-mediated catalysis. The germline KRAS point mutations found in Noonan syndrome encode different amino acid substitutions that have less severe biochemical consequences. Together, these observations and the structural conservation of the Ras/GAP switch through evolution suggest that a limited spectrum of mutations can constitutively activate Ras and cause disease. Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm characterized by driver Ras pathway mutations in 85% of cases, including common somatic KRAS and NRAS substitutions. We unexpectedly identified a novel somatic KRAS insertion, 66GQEEYSA67, in the bone marrow of a patient who meets most, but not all diagnostic criteria, for JMML and lacks any other Ras pathway mutation. This seven amino acid insertion is located in the conserved Switch II domain of Ras, which binds to and activates PI3 kinase and other Ras effectors. We characterized the functional and biochemical consequences of this mutation, and of a similar five amino acid insertion identified in two lung cancers and one colon cancer archived in the COSMIC database (66EEYSA67). We find that unlike WT K-Ras, the expression of both insertion mutants supports cytokine independent growth of IL-3 dependent pro-B Ba/F3 cells similar to oncogenic K-RasG12D. Ba/F3 cells expressing K-Ras66GQEEYSA67 or K-Ras66EEYSA67 mutant proteins have markedly elevated levels of Ras-GTP that are insensitive to IL-3 stimulation, and result in constitutive ERK and Akt phosphorylation. Confirming that these Switch II insertion mutants have oncogenic potential in the hematopoietic system, fetal liver cells transduced with KRas66GQEEYSA67, KRas66EEYSA67, or KRasG12D formed cytokine independent colony forming unit granulocyte macrophage (CFU-GM) colonies and also exhibited GM-CSF hypersensitivity, which is a cellular hallmark of JMML. Biochemical characterization of recombinant K-Ras66GQEEYSA67 and K-Ras66EEYSA67 proteins showed them to have reduced intrinsic GTP hydrolysis, and to accumulate in the GTP-bound conformation when compared to oncogenic K-RasG12D. Furthermore, whereas recombinant p120 GAP markedly accelerated the rate of GTP hydrolysis by WT K-Ras, K-Ras66GQEEYSA67 and K-Ras66EEYSA67 were completely resistant. The most likely structural explanation is that these insertions perturb critical contacts between glutamine 61 in the Switch II domain of Ras and GAPs. We conclude that KRAS66GQEEYSA67 and KRAS66EEYSA67 are oncogenic driver mutations in JMML and other cancers. These data illuminate a new class of oncogenic RAS mutations and are of immediate diagnostic relevance. Furthermore, these data reveal unanticipated structural flexibility in a domain of Ras that plays a critical role in effector binding, which has implications for potential mechanism of resistance to emerging therapeutic approaches. Disclosures Bollag: Plexxikon: Employment. Zhang:Plexxikon: Employment.
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