Ras Gene Mutations Research Articles

Abstract 326: Discovery and in vitro and in vivo characterization of a novel, small-molecule Ras inhibitor class

Abstract Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Through iterative screening and chemical optimization using a phenotypic assay designed to select for Ras inhibitors, we identified a novel series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras. Several compounds in the series have favorable drug-like properties with strong antitumor activity in a mouse K-Ras mutant tumor model. Methods: Viable cell number was measured using a luminescent indicator of ATP. Disruption of Ras-Raf binding was determined by pre-incubating GST-Raf beads with cell lysates in the presence of test compounds for 30 min. Ras activation was measured by Ras pull-down and western blotting using an anti-Ras antibody. Cell cycle arrest was measured by DNA content. Antitumor activity was determined in a subcutaneous mouse tumor model involving K-Ras mutant HCT116 colon tumors. Mice were treated with Ras inhibitors administered ip for 14 days bid at a dose of 2.5 or 5 mg/kg. Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines harboring activated K-Ras, N-Ras or H-Ras with a selectivity index greater than 100-fold over cells lacking activated Ras. Ras selectivity was confirmed by transfecting human HT29 colon and H322 lung tumor cells that lack activated Ras with mutant H-Ras, thus inducing sensitivity to DC070-547. The compound also inhibited Ras-Raf binding as evident by Ras-pull down assays of lysates from tumor cells treated with DC070-0547 at concentrations that inhibit tumor cell growth. DC070-547 caused G2 phase cell cycle arrest and induced apoptosis selectively in tumor cells containing activated Ras. Cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment.The Ras inhibitors were evaluated for antitumor activity in a subcutaneous mouse model involving K-Ras mutant human HCT116 colon tumors. Treatments were well tolerated and completely suppressed tumor growth with the effect being sustained for at least six weeks after treatment was discontinued. Complete tumor regression was also apparent in some of the treated mice. Conclusion: While Ras is widely considered to be non-druggable, we have identified a novel series of compounds that potently and selectivity inhibit the growth of tumor cells harboring activated Ras. With promising drug-like properties, these compounds represent a first in class series of Ras inhibitors from which several prospective drug development candidates have been identified.. These results support further preclinical development and future Phase I/II clinical evaluation in patients with Ras-driven cancers. Citation Format: Adam B. Keeton, Bing Zhu, Kevin J. Lee, Joshua C. Canzoneri, Sara C. Sigler, Ashley S. Lindsey, Veronica Ramirez-Alcantara, Luciana Barnes, Tyler E. Mattox, Kate McConnell, Kristy L. Berry, Jacob Valiyaveettil, Xi Chen, Michael R. Boyd, Gary A. Piazza. Discovery and in vitro and in vivo characterization of a novel, small-molecule Ras inhibitor class. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 326.

Troponin-I enhances and is required for oncogenic overgrowth.

Human tumors of various tissue origins show an intriguing over-expression of genes not considered oncogenes, such as that encoding Troponin-I (TnI), a well-known muscle protein. Out of the three TnI genes known in humans, the slow form, TNNI1, is affected the most. Drosophila has only one TnI gene, wupA. Here, we studied excess- and loss-of function of wupA in Drosophila, and assayed TNNI1 down regulation in human tumors growing in mice. Drosophila TnI excess-of-function increases proliferation and potentiates oncogenic mutations in Ras, Notch and Lgl genes. By contrast, TnI loss-of-function reduces proliferation and antagonizes the overgrowth due to these oncogenic mutations. Troponin-I defective cells undergo Flower- and Sparc-dependent cell competition. TnI can localize to the nucleus and its excess elicits transcriptional up-regulation of InR, Rap1 and Dilp8, which is consistent with the increased cell proliferation. Human tumor cell lines treated with a human Troponin-I peptide arrest in G0/G1. In addition, proliferation of non-small-cell lung carcinoma xenografts in mice is restrained by TNNI1 down-regulation. Thus, Troponin-I reveals a novel function in cell proliferation that may be of therapeutic interest in certain types of cancer.

Detection of Molecular Alterations in Medullary Thyroid Carcinoma Using Next-Generation Sequencing: an Institutional Experience.

Medullary thyroid carcinoma (MTC) harbors rearranged during transfection (RET) gene and rarely RAS gene mutations. The knowledge of the type of gene mutation in MTC is important to determine the treatment of the patients and the management of their family members. Targeted next-generation sequencing with a panel of 47 genes was performed in a total of 12 cases of sporadic (9/12) and hereditary MTC (3/12). Two of three hereditary MTCs had RET/C634R mutation, while the other one harbored two RET mutations (L790F and S649L). All the sporadic MTC had RET/M918T mutation except one case with HRAS mutation. Next-generation sequencing (NGS) can provide comprehensive analysis of molecular alterations in MTC in a routine clinical setting, which facilitate the management of the patient and the family members.

The opposite association of HRAS and KRAS mutations with clinical variables of bladder cancer

The HRAS, KRAS, and NRAS gene products belong to the superfamily of small GTPases. These proteins regulate the cellular response to extracellular stimuli through the activation of different signaling pathways. Although the role of the RAS gene mutations in the pathogenesis of various human cancers has been established, the clinical significance of these molecular alterations in bladder cancer remains unclear. The aim of this study was to determine the frequency and spectrum of the HRAS, KRAS, and NRAS mutations, to analyze their association with the clinicopathological variables, and to determine the prognostic value of these alterations in terms of recurrence, progression and mortality, in a prospective cohort of 249 bladder cancer patients. The frequency of the RAS mutations, detected by the SNaPshot method, was 11.2%, of which the HRAS mutations accounted for 64.3%, KRAS, for 28.6%, and NRAS, for 7.1%. We failed to find any correlation between all the RAS gene mutations and pathomorphological characteristics. However, when analyzed separately, the opposite association of the HRAS and KRAS mutations with the clinical parameters of bladder cancer was for the first time shown: the HRAS mutations were significantly associated with low stage low grade papillary tumors of a small size (p < 0.05), whereas the KRAS mutations were associated with non-papillary urothelial carcinomas and the presence of metastases (p < 0.05). Analysis of the prognostic value of the molecular alterations revealed the association of the KRAS mutations with decreased cancer-specific survival in both the total group of patients and the subgroup with nonmuscle invasive disease. The data obtained suggest that the HRAS and KRAS gene mutations may characterize alternative pathways of bladder cancer pathogenesis: the HRAS mutations indicating benign and KRAS mutations, aggressive disease course.

Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer.

The ability of Natural Killer (NK) cells to kill tumor targets has been extensively studied in various hematological malignancies. However, NK cell therapy directed against solid tumors is still in early development. Epidermal Growth Factor Receptor (EGFR) targeted therapies using monoclonal antibodies (mAbs) such as cetuximab and panitumumab are widely used for the treatment of metastatic colorectal cancer (mCRC). Still, the clinical efficacy of this treatment is hampered by mutations in RAS gene, allowing tumors to escape from anti-EGFR mAb therapy. It is well established that NK cells kill tumor cells by natural cytotoxicity and can in addition be activated upon binding of IgG1 mAbs through Fc receptors (CD16/FcγRIIIa) on their surface, thereby mediating antibody dependent cellular cytotoxicity (ADCC). In the current study, activated Peripheral Blood NK cells (PBNK) were combined with anti-EGFR mAbs to study their effect on the killing of EGFR+/- cancer cell lines, including those with RAS mutations. In vitro cytotoxicity experiments using colon cancer primary tumors and cell lines COLO320, Caco-2, SW620, SW480 and HT-29, demonstrated that PBNK cells are cytotoxic for a range of tumor cells, regardless of EGFR, RAS or BRAF status and at low E:T ratios. Cetuximab enhanced the cytotoxic activity of NK cells on EGFR+ tumor cells (either RASwt, RASmut or BRAFmut) in a CD16 dependent manner, whereas it could not increase the killing of EGFR- COLO320. Our study provides a rationale to strengthen NK cell immunotherapy through a combination with cetuximab for RAS and BRAF mutant mCRC patients.

Somatic mutation profiling of follicular thyroid cancer by next generation sequencing

The molecular etiology of follicular thyroid tumors is largely unknown, rendering the diagnostics of these tumors challenging. The somatic alterations present in these tumors apart from RAS gene mutations and PAX8/PPARG translocations are not well described. To evaluate the profile of somatic alteration in follicular thyroid tumors, a total of 82 thyroid tissue samples derived from 48 patients were subjected to targeted Illumina HiSeq next generation sequencing of 372 cancer-related genes. New somatic alterations were identified in oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK). Single nucleotide and large structural variants were most and least frequently identified, respectively. A novel translocation in DERL/COX6C was detected. Many somatic alterations in non-coding gene regions with high penetrance were observed. Thus, follicular thyroid tumor somatic alterations exhibit complex patterns. Most tumors contained distinct somatic alterations, suggesting previously unreported heterogeneity.

RAS isoforms and mutations in cancer at a glance.

RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts.

Molecular mechanisms of transformation from actinic keratosis to squamous cell carcinoma

Actinic keratosis (AK) is a precursor to cutaneous squamous cell carcinoma (SCC) , and molecular mechanisms of transformation from AK to SCC have been a research hotspot. This review focuses on four aspects of the molecular mechanisms, including chromosomal variation, gene mutations, signaling pathways and other factors, summarizes advances in selective mutations in chromosomes 3p, 9p, 9q, 13q, 17p and 17q, heterozygous deletion mutations in chromosome 18q, mutations of p53, p16 and Ras genes, abnormality of the transforming growth factor β1 signaling pathway and Fas/FasL signaling pathway, and aberrant expression of matrix metalloproteinases in the transformation from AK to SCC, which will provide a reference for further researches. Key words: Keratosis, actinic; Neoplasms, squamous cell; Genes; Mutation; Signal transduction; Genetic variation

OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination.

Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.

Mutation in BRAF and Other Members of the MAPK Pathway in Papillary Thyroid Carcinoma in the Pediatric Population.

Papillary thyroid carcinoma (PTC) is an uncommon tumor in the pediatric population. A limited number of studies have examined genetic mutations affecting the mitogen-activated protein kinase (MAPK) pathway in the pediatric population. To examine mutations affecting this pathway in PTC in our pediatric population and compare the BRAF V600E mutation rates in pediatric and adult tumors. Eighty-four patients, including 14 pediatric and 70 adult, with PTC were tested for the BRAF V600E mutation by using real-time polymerase chain reaction and sequencing. Additionally, we examined the rate of RAS point mutations with real-time polymerase chain reaction and rearrangements of RET/PTC1 and RET/PTC3 in the pediatric group with fluorescence in situ hybridization. Clinical and histologic data were compared as well. Of 77 tumors that had an interpretable result, the BRAF V600E mutant was identified in 4 of 13 pediatric patients (31%) and 43 of 64 adult patients (67%), which was a significant difference (using Fisher exact test, P = .03). One pediatric and 6 adult cases did not reveal an interpretable result with melting curve analysis. One of these cases harbored a rare 3-base pair deletion mutation (c.1799_1801delTGA). Mutations in RAS genes were not seen in any pediatric tumors. One tumor with a RET/PTC1 rearrangement and another with RET/PTC3 were identified in the pediatric population (15%). The rate of the BRAF V600E mutation in the pediatric population is significantly lower than that seen in the adult population. Mutations in RAS do not contribute significantly to pediatric PTC. This experience from our institution adds to the growing body of knowledge regarding tumor genetics in pediatric PTC.

Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR

ObjectiveTo validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of...

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine.

Detection of Novel Pathogenic Gene Rearrangements in Pediatric Acute Myeloid Leukemia By RNA Sequencing

BackgroundPediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia cases. AML is a major therapeutic challenge in pediatric oncology, and the current overall survival rate is <70%. The pathogenesis of AML is heterogeneous, and causes include various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expression, resulting in increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations such as t(8;21), inv(16), and MLL -rearrangements are well established as diagnostic and prognostic markers in AML. Furthermore, recurrent mutations in FLT3, KIT, and RAS have been reported in both adult and pediatric AML. Recently, massively parallel sequencing has facilitated the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for predicting the prognosis. However, these mutations are rare in pediatric AML, thereby suggesting that other genetic alterations may exist in pediatric AML.In addition, recent studies have reported that the NUP98-NSD1 fusion is an adverse AML prognostic marker and that PRDM16 (also termed MEL1) is a representative gene that is overexpressed in patients who have the NUP98-NSD1 fusion. PRDM16 overexpression occurs in nearly a quarter of pediatric AML patients who are NUP98-NSD1 negative, and this overexpression is increased in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1,and MLL-PTD).Patients and MethodsTo obtain a complete overview of gene rearrangements and other genetic lesions, we performed RNA sequencing of samples from 47 de novo pediatric AML patients using Illumina HiSeq 2000, including 39 patients with normal karyotypes and 6 patients with Trisomy 8. Among these 47 patients, 35 patients overexpressed PRDM16, which was strongly associated with a poor prognosis in our previous studies. As a control, we selected 12 patients with low PRDM16 expression levels. All patients were enrolled and treated as part of the AML-05 study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group. We determined the known gene mutations present in these patients using the RNA sequencing data.ResultsApproximately 300 candidate gene rearrangements were identified in 46/47 samples, including 26 in-frame and 78 out-of-frame gene rearrangements. Several recurrent gene rearrangements identified in this study involved previously reported targets in AML, such as FUS-ERG, NUP98-NSD1,and MLL-MLLT3. However, several novel gene rearrangements were identified in the current study, including HOXA10-HOXA-AS3, PRDM16-XXX, CUL1-YYY, and DAZAP1-ZZZ. At present, we are validating these novel gene rearrangements using Sanger sequencing. Known gene alterations, such as FLT3-ITD, MLL-PTD, and mutations of RAS, KIT, CEBPA, WT1, and NPM1 genes, were detected by RNA sequencing.ConclusionIn the present study, RNA sequencing was employed to elucidate the complexity of gene rearrangements/mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that can be discriminated from adult AML in terms of the spectrum of gene rearrangements/mutations. We identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including various novel fusion genes. Thus, our results suggest that gene rearrangements and mutations play essential roles in pediatric AML. DisclosuresNo relevant conflicts of interest to declare.

Fusion Transcript Reduction in Core Binding Factor Acute Myeloid Leukemia: Maintenance Strategy with Hypomethylating Agents

Introduction: Recurrent translocations, t(8;21) or inv(16), in core binding factor acute myeloid leukemia (CBF-AML) are amenable to monitoring for minimal residual disease (MRD) with reverse transcriptase polymerase chain reaction (RTPCR). Fusion products of these translocations recruit epigenetic silencing complexes resulting in hematopoietic maturation arrest. Despite a favorable prognosis, disease relapse remains the single cause of treatment failure in CBF-AML. We hypothesized that maintenance therapy with hypomethylating agents (HMA), including decitabine (DAC) and azacytidine (AZA), can be used for MRD elimination to ultimately prolong relapse free survival.Methods: We performed a retrospective analysis of 23 patients with CBF-AML who received HMA after fludarabine, cytarabine and G-CSF (FLAG) with low dose gemtuzumab or idarubicin induction/consolidation. RTPCR from peripheral blood or bone marrow was obtained approximately every 3 months. Mutations in KIT, FLT3ITD, FLT3D835 and RAS genes were tested at baseline.Results: A total of 23 patients [t(8;21)=15 and inv(16)=8] received maintenance HMA. The reason for transition to HMA was persistent PCR positivity in 13 patients (57%), prolonged myelosuppression precluding chemotherapy based consolidation in 9 patients (39%), and molecular relapse after allogeneic stem cell transplant (SCT) in 1 patient (4%). Three of these patients received salvage clofarabine, cytarabine and idarubicin (CIA) for relapsed CBF-AML after FLAG based therapy and then went on HMA for molecular MRD. Of 23 patients, 21 received DAC and 2 received AZA. Median age at diagnosis was 53 (range, 23-70). Patients received a median of 6 cycles of FLAG based induction/consolidation (range, 1-7) prior to transitioning to HMA. The median number of HMA cycles received was 6 (range, 1-17). Seventeen patients had detectable MRD (RTPCR ≥ 0.01) at initiation of HMA maintenance with a median RTPCR of 0.06 (range, 0.03-0.91). All 17 patients were in morphologic and cytogenetic complete remission (CR) when HMA was initiated. Of these 17 patients, 5 patients (29%) had progressive disease post HMA initiation, including 1/7 with t(8:21) and 4/10 with inv(16). The RTPCR values preceding HMA in these 5 patients were 0.34, 0.06, 0.08, 0.01, and 0.01, respectively (median=0.06). All 5 patients had an increase in the RTPCR value soon after the initiation of HMA. RTPCR for these 5 patients following the first or second cycle of HMA was 3.84, >100, 77.48, 1.33, and 0.17, respectively. Four [inv(16)=3 and t(8;21)=1] of these 5 patients had a RAS mutation, while the patient with t(8;21) had a concomitant KIT mutation, and another patient with inv(16) had a FLT3D835 mutation. All five patients failing HMA proceeded to SCT, and 3 are alive and disease free post SCT. For the 12 patients without HMA failure, the median RTPCR at HMA initiation was 0.06 (range, 0.01-0.91). Unlike the HMA failure subset described above, 11 of these patients had a reduction in RTPCR by first or second subsequent measurement following initiation of HMA. One patient is in early follow up. All 6 patients who started HMA with undetectable MRD remain MRD negative (RTPCR < 0.01) and in remission. Eight patients continue on maintenance therapy. Median follow up is 11.3 months (range, 2.9-67.7).Conclusions: Despite a favorable prognosis in CBF-AML, relapse remains the primary cause of treatment failure. Identifying patients who are more likely to relapse will be beneficial in extending relapse free survival. Hypomethylating agents can potentially be used in patients with detectable RTPCR to prolong remission. Determining which patients would benefit most from this therapy is an important task. This data suggests that patients with low levels of RTPCR (between 0.01 and 0.05) following induction/consolidation chemotherapy might benefit most from maintenance HMA, particularly those that have a reduction in RTPCR on first or second subsequent measurement following initiation of hypomethylating agent therapy. DisclosuresDaver:ImmunoGen: Other: clinical trial, Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria.

The Deubiquitylase Usp22 Is Important for KRAS-Driven Myeloproliferative Disease

Mutations in Ras pathway are among the most common gene abnormalities in several types of cancer including hematological malignances. Ras gene (NRAS or KRAS) mutations are found in 20-40% of myeloid malignances and in 15-30% of acute lymphoblastic leukemia. Mutations in KRAS are the second most frequently mutated. Expression of the most common KRAS mutation (G12D) in mouse hematopoietic cells causes a fatal myeloproliferative neoplasm (MPN). The disease observed in these mice resembles the human MPNs juvenileand chronic myelomonocytic leukemias characterized by leukocytosis, splenomegaly and anemia. Patients with these MPNs typically survive for 10 months to 4 years and are still considered incurable.Previous studies comparing mouse and human samples led to the identification of USP22 (Ubiquitin Specific Peptidase 22) as part of a self-renewal gene expression signature in different types of cancer including hematological malignances. We analyzed the publicly available database, Oncomine, and showed that USP22 is highly expressed in acute myeloid and lymphoid leukemias. Moreover, USP22 was found as part of an oncogenic network in acute myeloid leukemia with mutations in an upstream activator of Ras signaling. Thus, to study a possible role of Usp22 in Ras-induced myeloproliferative disease, we generated KrasG12D/+ Mx1Cre/+ (hereby named KM), KrasG12D/+ Mx1Cre/+ Usp22F/F (KMU) and Mx1Cre/+ Usp22F/F (MU) mice.Analysis of peripheral blood cells showed no changes in blood cell counts in MU mice compared to those of control mice, indicating that Usp22 deletion does not affect steady state hematopoiesis. As expected, KM mice had an increase in neutrophils and monocytes as previously reported. In contrast, KMU mice showed a dramatic decrease in neutrophil counts, suggesting that USP22 is possibly involved in KRAS-induced myeloproliferative disease.Analysis of bone marrow populations showed no significant changes in the frequency of HSCs, GMP, CMP or MEP populations between KM and KMU mice. However, there was a statistically significant decrease in the Gr1+ CD11b+ population in KMU compared to KM mice. This decrease was mostly associated with a decrease in Gr1 expression and accumulation of CD11b+ cells. We also performed studies using colony-forming assays in methylcellulose media. Bone marrow cells from KM mice were able to form colonies in the absence of cytokines as previously reported. However, Usp22 deletion (KMU mice) decreased cytokine-independent growth by more than 50%.Previous reports have shown that transplantation of KM bone marrow cells lead to the generation of myeloproliferative disease and thymic lymphomas in recipient mice. To investigate whether Usp22 deletion could limit the generation of these malignances, we transplanted bone marrow cells from KM and KMU mice into lethally irradiated mice. Although KMU recipient mice died earlier than KM recipient mice for unknown reasons, myeloproliferative disease was observed in only 25% of KMU recipient mice compared to 75% of KM recipient mice. Moreover, the thymus size of KMU recipient mice was statistically significant smaller than those of KM recipient mice.In summary, we found that Usp22 may play an important role in Kras-driven myeloid and lymphoid malignances. Usp22 deletion in KM mice induced a dramatic decrease in neutrophil counts, decreased numbers of Gr1+ CD11b+ cells in the bone marrow and decreased cytokine-independent growth of KM bone marrow cells. Moreover, transplantation of KMU cells into lethally irradiated recipients resulted in decreased generation of myeloid and lymphoid malignances. We are currently performing additional studies to dissect the molecular mechanism by which Usp22 affects Kras-driven hematological malignances. DisclosuresNo relevant conflicts of interest to declare.

The opposite association of HRAS and KRAS mutations with clinical variables of bladder cancer

HRAS, KRAS and NRAS gene products belong to the superfamily of small GTPases. These proteins regulate cellular response to extracellular stimuli by means of activation of different signaling pathways. Although the role of RAS gene mutations in the pathogenesis of various human cancers has been established, the clinical significance of these molecular alterations in bladder cancer remains unclear. The aim of this study was to determine the frequency and spectrum of HRAS, KRAS and NRAS mutations, to analyze their relationships with clinicopathological variables and to determine the prognostic value of these alterations in terms of recurrence, progression and mortality, in a prospective cohort of 249 bladder cancer patients. The frequency of RAS mutations detected by the SNaPshot method, was found to be 11.2 %, of which HRAS mutations accounted for 64.3 %, KRAS, for 28.6 % and NRAS, for 7.1 %. We failed to find any correlation between all RAS mutations and pathomorphological characteristics. However, when analyzed separately, HRAS and KRAS mutations were for the first time shown to be associated with the opposite clinical parameters of bladder cancer: HRAS mutations were significantly associated with low-stage low-grade papillary tumors of a small size (р &lt; 0.05), whereas KRAS mutations were associated with non-papillary urothelial carcinomas and the presence of metastasis (р &lt; 0.05). Analysis of the prognostic value of molecular alterations revealed an association of KRAS mutations with decreased cancer-specific survival in both the whole group of patients and the subgroup with non-muscle invasive disease. The data obtained suggest that HRAS and KRAS gene mutations may characterize alternative pathways of bladder cancer pathogenesis: HRAS mutations indicating benign and KRAS mutations, aggressive disease course.

Abstract A140: Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras

Abstract Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Through iterative chemical synthesis and screening using a phenotypic assay designed to select for Ras inhibitors, we identified a small-molecule lead compound that was then optimized for potency and selectivity to inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras. Materials and methods: Viable cell number was measured using the CellTiter-Glo® ATP assay (Promega) following 72 hr of treatment. Ras activation status was measured by precipitating GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose followed by western blotting using anti-Ras antibody. Ras binding was determined by pre-incubating cell lysates with GST-Raf1-RBD/GSH sepharose, treating with test compounds for 30 min, followed by western blotting using anti-Ras antibody. Cell cycle distribution was measured by DNA content following DyeCycle Green labeling. Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines harboring activated K-Ras, N-Ras or H-Ras with selectivity indices greater than 100-fold over cells lacking activated Ras. By surveying a large panel of human colon, breast, and lung tumor cell lines, a strong correlation was measured between potency to inhibit tumor cell growth and Ras activation status. Ras selectivity was confirmed by transfecting human H322 lung tumor cells that lack activated Ras with mutant H-Ras and inducing sensitivity to DC070-547. The compound inhibited Ras-Raf binding as shown by Ras-pull down in cell lysates following treatment with DC070-547 at concentrations that inhibit tumor cell growth. DC070-547 also caused cell cycle arrest in the G2 phase selectively in tumor cells containing activated Ras. Interestingly, cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment. Conclusion: While Ras is widely considered to be non-druggable, we have identified a series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras. With promising drug-like properties, DC070-547 has been selected from this series as a prospective drug development candidate that is being evaluated for anti-tumor efficacy and toxicity in preclinical models. Citation Format: Gary A. Piazza, Bing Zhu, Kevin Lee, Joshua Canzoneri, Sara Sigler, Ashley Lindsey, Veronica Ramirez-Alcantara, Luciana Madeira da Silva, Haddon Mullins, Alisa Trinh, Kristy Berry, Jacob Valiyaveettil, Adam B. Keeton, Xi Chen, Michael R. Boyd. Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A140.

Genetic Alterations in Differentiated Thyroid Cancer Patients with Acromegaly.

Acromegaly is associated with increased thyroid cancer risk. We aimed to analyze the frequency of point mutations of BRAF and RAS genes, and RET/PTC, PAX8/PPARγ gene rearrangements in patients with acromegaly having differentiated thyroid cancers (DTC) and their relation with clinical and histological features. 14 acromegalic patients (8 male, 6 female) with DTC were included. BRAF V600E and NRAS codon 61 point mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ gene rearrangements were analyzed in thyroidectomy specimens. We selected 14 non-acromegalic patients with DTC as a control group. 2 patients (14.3%) were detected to have positive BRAF V600E and 3 patients (21.4%) were detected to have NRAS codon 61 mutation. NRAS codon 61 was the most frequent genetic alteration. Patients with positive mutation had aggressive histologic features more frequently than patients without mutations. Comparison of the acromegalic and non-acromegalic patients with DTC revealed that BRAF V600E mutation was more frequent in non-acromegalic patients with DTC (14.2% vs. 64.3%, p=0.02). RET/PTC 1/ 3, PAX8/PPARγ gene rearrangements were not detected in any patient. None of the patients including the patients with positive point mutations had recurrence, and local and/or distant metastasis. NRAS codon 61 is the most frequent genetic alteration in this acromegaly series with DTC. Since acromegalic patients have lower prevalance of BRAF V600E mutation, BRAF V600E mutation may not be a causative factor in development of DTC in acromegaly. Despite the relation of BRAF V600E and NRAS codon 61 mutations with aggresive histopathologic features, their impact on tumor prognosis remains to be defined in acromegaly in further studies.

RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity.

Extended RAS Gene Mutation Testing in Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015 Summary.

Purpose An American Society of Clinical Oncology Provisional Clinical Opinion (PCO) offers timely clinical direction after publication or presentation of potentially practice-changing data from major studies. This PCO update addresses the utility of extended RAS gene mutation testing in patients with metastatic colorectal cancer (mCRC) to detect resistance to anti–epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) therapy. Clinical Context Recent results from phase II and III clinical trials in mCRC demonstrate that patients whose tumors harbor RAS mutations in exons 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146) are unlikely to benefit from therapy with MoAbs directed against EGFR, when used as monotherapy or combined with chemotherapy.