Now showing items 1-20 of 2404

    • Distinct segments within the enhancer region collaborate to specify the type of leukemia induced by nondefective Friend and Moloney viruses

      Fox Chase Cancer Center (Temple University) (1989-01-01)
      The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)
    • Disease specificity of nondefective Friend and Moloney murine leukemia viruses is controlled by a small number of nucleotides

      Fox Chase Cancer Center (Temple University) (1987-03-01)
      Moloney murine leukemia virus induces T cell lymphomas after injection into NFS mice, whereas the nondefective Friend virus induces erythroleukemias. Previous studies showed that sequences encompassing the viral transcriptional signals in U3 are the primary determinant of this phenotype in recombinants between these two viruses. To more precisely identify the sequences responsible, we constructed additional recombinants, within U3, between Friend and Moloney viruses and assayed these recombinants for for their disease specificity. We found that a fragment 191 bases long that included the direct repeat (enhancer) region plus 22 nucleotides to its 3' side from Friend virus was sufficient to convert Moloney virus to a virus that induced only erythroleukemias. A 171-base-long fragment of Moloney virus, including just the direct repeat, converted Friend virus to a virus that induced primarily lymphomas (about 85% of mice injected). We also constructed Moloney and Friend virus variants with one rather than two copies of the enhancer element. These viruses retained their disease specificity, although they exhibited a marked increase in the latent period of disease induction. Together the results suggest that 25 or fewer nucleotide differences, lying within and also just 3' of the direct repeat, are the primary determinant of the distinct disease specificities of nondefective Friend and Moloney viruses.
    • Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design

      Fox Chase Cancer Center (Temple University) (1990-02-01)
      We aligned published sequences for the U3 region of 35 type C mammalian retroviruses. The alignment reveals that certain sequence motifs within the U3 region are strikingly conserved. A number of these motifs correspond to previously identified sites. In particular, we found that the enhancer region of most of the viruses examined contains a binding site for leukemia virus factor b, a viral corelike element, the consensus motif for nuclear factor 1, and the glucocorticoid response element. Most viruses containing more than one copy of enhancer sequences include these binding sites in both copies of the repeat. We consider this set of binding sites to constitute a framework for the enhancers of this set of viruses. Other highly conserved motifs in the U3 region include the retrovirus inverted repeat sequence, a negative regulatory element, and the CCAAT and TATA boxes. In addition, we identified two novel motifs in the promoter region that were exceptionally highly conserved but have not been previously described.
    • Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology

      Fox Chase Cancer Center (Temple University) (1995-07-01)
      Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.
    • Mutation of the core or adjacent LVb elements of the Moloney murine leukemia virus enhancer alters disease specificity

      Fox Chase Cancer Center (Temple University) (1990)
      Transcriptional enhancers of replication-competent mouse C-type retroviruses are potent determinants of the distinct disease-inducing phenotypes of different viral isolates and can also strongly influence the incidence and latent period of disease induction. To study the contribution of individual protein-binding sites to viral pathogenicity, we introduced mutations into each of the known nuclear factor-binding sites in the enhancer region of the Moloney murine leukemia virus and injected viruses with these mutations into newborn NFS mice. All viruses induced disease. Viruses with mutations in both copies of the leukemia virus factor a (LVa) site, leukemia virus factor c (LVc) site, or in just the promoter proximal copy of the glucocorticoid response element (GRE) had a latent period of disease onset and disease specificity indistinguishable from that of the wild-type Moloney virus. Viruses with mutations in two or three of the GREs, in both copies of the leukemia virus factor b (LVb) site, in two of the four nuclear factor 1 (NF1) consensus motifs, or in both copies of the conserved viral core element showed a significant delay in latent period of disease induction. Strikingly, viruses with mutations in the core element induced primarily erythroleukemias, and mutations in the LVb site also resulted in a significant incidence of erythroleukemias. These and other genetic and biochemical studies suggest models for how subtle alterations in the highly conserved structure of mouse C-type retrovirus enhancers can produce a dramatic effect on disease specificity.
    • HEF1-Aurora A Interactions

      Fox Chase Cancer Center (Temple University) (2006-01-18)
      Regulated timing of cell division cycles, and geometrical precision in the planar orien-tation of cell division, are critical during organismal development and remain important for the maintenance of polarized structures in adults. Mounting evidence suggests that these processes are coordinated at the centrosome through the action of proteins that mediate both cell cycle and cell attachment. Our recent work identifying HEF1 as anactivator of the Aurora A kinase suggests a novel hub for such integrated signaling. Wesuggest that defects in components of the machinery specifying the temporal and spatial integration of cell division may induce cancer and other diseases through pleiotropic effects on cell migration, proliferation, apoptosis, and genomic stability.
    • The Related Adhesion Focal Tyrosine Kinase Differentially Phosphorylates p130Cas and the Cas-like Protein, p105HEF1

      Fox Chase Cancer Center (Temple University) (1997-08-08)
      The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following β1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130Cas and HEF1, phosphorylation of p130Cas, but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130Cas and HEF1, but only the C-terminal domain of p130Cas. Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130Cas. These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130Cas.
    • The Tuberous Sclerosis 2 Gene Product, Tuberin, Functions as a Rab5 GTPase Activating Protein (GAP) in Modulating Endocytosis

      Fox Chase Cancer Center (Temple University) (1997-03-07)
      The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This “suppressor− activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis.
    • The Ankyrin Repeat-containing Adaptor Protein Tvl-1 Is a Novel Substrate and Regulator of Raf-1

      Fox Chase Cancer Center (Temple University) (1999-05-21)
      Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro andin vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream ofMEF2B with the two genes in a head to head arrangement.
    • A Novel Cas Family Member, HEPL, Regulates FAK and Cell Spreading

      Fox Chase Cancer Center (Temple University) (2008-02-06)
      For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.
    • TACCing on new functions for the TSC2 tumor suppressor

      Fox Chase Cancer Center (Temple University) (2010-04-01)
    • DUSP6 regulates drug sensitivity by modulating DNA damage response

      Fox Chase Cancer Center (Temple University) (2013-07-09)
      Background: Dual specificity phosphatase 6 (DUSP6) is a member of a family of mitogen-activated protein kinase phosphatases that dephosphorylates and inhibits activated ERK1/2. Dual specificity phosphatase 6 is dynamically regulated in developmental and pathological conditions such as cancer. Methods: Cancer cell lines were made deficient in DUSP6 by siRNA and shRNA silencing. Sensitivity to anti-EGFR and chemotherapeutic agents was determined in viability and apoptosis assays, and in xenografts established in SCID mice. Cellular effects of DUSP6 inactivation were analysed by proteomic methods, followed by analysis of markers of DNA damage response (DDR) and cell cycle. Results: We determined that depletion of DUSP6 reduced the viability of cancer cell lines and increased the cytotoxicity of EGFR and other targeted inhibitors, and cytotoxic agents, in vitro and in vivo. Subsequent phosphoproteomic analysis indicated DUSP6 depletion significantly activated CHEK2 and p38, which function in the DDR pathway, and elevated levels of phosphorylated H2AX, ATM, and CHEK2, for the first time identifying a role for DUSP6 in regulating DDR. Conclusion: Our results provide a novel insight into the DUSP6 function in regulating genomic integrity and sensitivity to chemotherapy in cancer.
    • Opposing effects of inhibitors of Aurora-A and EGFR in autosomal-dominant polycystic kidney disease

      Fox Chase Cancer Center (Temple University) (2015-10-16)
      Aurora-A kinase (AURKA) overexpression in numerous tumors induces aneuploidy, in part because of cytokinetic defects. Alisertib and other small-molecule inhibitors targeting AURKA are effective in some patients as monotherapies or combination therapies. Epidermal growth factor receptor (EGFR) pro-proliferative signaling activity is commonly elevated in cancer, and the EGFR inhibitor erlotinib is commonly used as a standard of care agent for cancer. An erlotinib/alisertib combination therapy is currently under assessment in clinical trials, following pre-clinical studies that indicated synergy of these drugs in cancer. We were interested in further exploring the activity of this drug combination. Beyond well-established functions for AURKA in mitotic progression, additional non-mitotic AURKA functions include control of ciliary stability and calcium signaling. Interestingly, alisertib exacerbates the disease phenotype in mouse models for autosomal-dominant polycystic kidney disease (ADPKD), a common inherited syndrome induced by aberrant signaling from PKD1 and PKD2, cilia-localized proteins that have calcium channel activity. EGFR is also more active in ADPKD, making erlotinib also of potential interest in this disease setting. In this study, we have explored the interaction of alisertib and erlotinib in an ADPKD model. These experiments indicated erlotinib-­restrained cystogenesis, opposing alisertib action. Erlotinib also interacted with alisertib to regulate proliferative signaling proteins, albeit in a complicated manner. Results suggest a nuanced role of AURKA signaling in different pathogenic conditions and inform the clinical use of AURKA inhibitors in cancer patients with comorbidities.
    • TumorNext: A comprehensive tumor profiling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy

      Fox Chase Cancer Center (Temple University) (2016-09-08)
      The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the “second hit” in hereditary cancer patients.
    • Interaction of germline variants in a family with a history of early-onset clear cell renal cell carcinoma

      Fox Chase Cancer Center (Temple University) (2019-01-24)
      Background: Identification of genetic factors causing predisposition to renal cell carcinoma has helped improve screening, early detection, and patient survival. Methods: We report the characterization of a proband with renal and thyroid cancers and a family history of renal and other cancers by whole-exome sequencing (WES), coupled with WES analysis of germline DNA from additional affected and unaffected family members. Results: This work identified multiple predicted protein-damaging variants relevant to the pattern of inherited cancer risk. Among these, the proband and an affected brother each had a heterozygous Ala45Thr variant in SDHA, a component of the succinate dehydrogenase (SDH) complex. SDH defects are associated with mitochondrial disorders and risk for various cancers; immunochemical analysis indicated loss of SDHB protein expression in the patient’s tumor, compatible with SDH deficiency. Integrated analysis of public databases and structural predictions indicated that the two affected individuals also had additional variants in genes including TGFB2, TRAP1, PARP1, and EGF, each potentially relevant to cancer risk alone or in conjunction with the SDHA variant. In addition, allelic imbalances of PARP1 and TGFB2 were detected in the tumor of the proband. Conclusion: Together, these data suggest the possibility of risk associated with interaction of two or more variants.
    • PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization

      Fels Institute for Cancer Research and Molecular Biology (Temple University); Fox Chase Cancer Center (Temple University) (2019-12-10)
      The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.
    • Targeted delivery of chemotherapy using HSP90 inhibitor drug conjugates is highly active against pancreatic cancer models

      Fox Chase Cancer Center (Temple University) (2016-10-13)
      The lack of effective treatment modalities is a major problem in pancreatic cancer (PCa), a devastating malignancy that is nearly universally driven by the “undruggable” KRAS and TP53 cancer genes. Poor tumor tissue penetration is the major source of resistance in pancreatic cancer where chemotherapy is the mainstay of treatment. In this study we exploited the selective tumor-targeting properties of the heat shock 90 protein inhibitors as the vehicle for drug delivery to pancreatic tumor tissues. STA-12-8666 is a novel esterase-cleavable conjugate of an HSP90i and a topoisomerase I inhibitor, SN-38. STA-12-8666 selectively binds activated HSP90 and releases its cytotoxic payload resulting in drug accumulation in pancreatic cancer cells in vivo. We investigated the preclinical activity of STA-12-8666 in patient derived xenograft and genetic models of pancreatic cancer. Treatment with STA-12-8666 of the KPC mice (knock-in alleles of LSL-KrasG12D, Tp53fl/fl and Pdx1-Cre transgene) at the advanced stages of pancreatic tumors doubled their survival (49 days vs. 74 days, p=0.008). STA-12-8666 also demonstrated dramatically superior activity in comparison to equimolar doses of irinotecan against 5 patient-derived pancreatic adenocarcinoma xenografts with prolonged remissions in some tumors. Analysis of activity of STA-12-8666 against tumor tissues and matched cell lines demonstrated prolonged accumulation and release of cytotoxic payload in the tumor leading to DNA damage response and cell cycle arrest. Our results provide a proof-of-principle validation that HSP90i-based drug conjugates can overcome the notorious treatment resistance by utilizing the inherently high affinity of pancreatic cancer cells to HSP90 antagonists.
    • Musashi-2 (MSI2) regulation of DNA damage response in lung cancer

      Fox Chase Cancer Center (Temple University) (2023-06-14)
      Lung cancer is one of the most common types of cancers worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras-activating mutation and Trp53 deletion, with and without Msi2 deletion (KP versus KPM2 mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice what supports published data. In addition, using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM/Atm mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo. Taken together, we conclude that MSI2 supports lung tumorigenesis, in part, by direct positive regulation of ATM protein expression and DDR. This adds the knowledge of MSI2 function in lung cancer development. Targeting MSI2 may be a promising strategy to treat lung cancer.
    • Comprehensive characterization of RAS mutations in colon and rectal cancers in old and young patients

      Fox Chase Cancer Center (Temple University) (2019-08-19)
      Colorectal cancer (CRC) is increasingly appreciated as a heterogeneous disease, with factors such as microsatellite instability (MSI), cancer subsite within the colon versus rectum, and age of diagnosis associated with specific disease course and therapeutic response. Activating oncogenic mutations in KRAS and NRAS are common in CRC, driving tumor progression and influencing efficacy of both cytotoxic and targeted therapies. The RAS mutational spectrum differs substantially between tumors arising from distinct tissues. Structure-function analysis of relatively common somatic RAS mutations in G12, Q61, and other codons is characterized by differing potency and modes of action. Here we show the mutational profile of KRAS, NRAS, and the less common HRAS in 13,336 CRC tumors, comparing the frequency of specific mutations based on age of diagnosis, MSI status, and colon versus rectum subsite. We identify mutation hotspots, and unexpected differences in mutation spectrum, based on these clinical parameters.