mutational activation
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2022 ◽  
Author(s):  
Frank Hidalgo ◽  
Laura M Nocka ◽  
Neel H Shah ◽  
Kent Gorday ◽  
Naomi R Latorraca ◽  
...  

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H?Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H?Ras in mammalian Ba/F3 cells correlate well with results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase?activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g., at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen?deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with the destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Afonso R. M. Almeida ◽  
João L. Neto ◽  
Ana Cachucho ◽  
Mayara Euzébio ◽  
Xiangyu Meng ◽  
...  

AbstractInterleukin-7 receptor α (encoded by IL7R) is essential for lymphoid development. Whether acute lymphoblastic leukemia (ALL)-related IL7R gain-of-function mutations can trigger leukemogenesis remains unclear. Here, we demonstrate that lymphoid-restricted mutant IL7R, expressed at physiological levels in conditional knock-in mice, establishes a pre-leukemic stage in which B-cell precursors display self-renewal ability, initiating leukemia resembling PAX5 P80R or Ph-like human B-ALL. Full transformation associates with transcriptional upregulation of oncogenes such as Myc or Bcl2, downregulation of tumor suppressors such as Ikzf1 or Arid2, and major IL-7R signaling upregulation (involving JAK/STAT5 and PI3K/mTOR), required for leukemia cell viability. Accordingly, maximal signaling drives full penetrance and early leukemia onset in homozygous IL7R mutant animals. Notably, we identify 2 transcriptional subgroups in mouse and human Ph-like ALL, and show that dactolisib and sphingosine-kinase inhibitors are potential treatment avenues for IL-7R-related cases. Our model, a resource to explore the pathophysiology and therapeutic vulnerabilities of B-ALL, demonstrates that IL7R can initiate this malignancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2220-2220
Author(s):  
Robert L. Bowman ◽  
Tanmay Mishra ◽  
Shira E. Eisman ◽  
Louise Cai ◽  
Wenbin Xiao ◽  
...  

Abstract Genomic studies in acute myeloid leukemia (AML) have generated a near complete catalogue of genes mutated at varying frequencies both across patients and in individual leukemias. The high variability of mutation burden within a given leukemia is suggestive of a stepwise evolutionary process composed of early, clonal, mutations and subsequent subclonal events. The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in AML, with mutations frequently manifesting as internal tandem duplications (ITDs) in the juxtamembrane domain leading to constitutive kinase activation. Although FLT3 is commonly a subclonal mutational event, FLT3 ITD mutations portend a poor prognosis particularly when combined with DNMT3A and NPM1, earlier mutations that drive clonal expansion. Notwithstanding its role as a subclonal driver, previous preclinical FLT3 models have utilized retroviral overexpression or germline mutant expression at the endogenous locus precluding accurate temporal modeling of disease. These efforts have prohibited evaluation of FLT3 mutational acquisition in the context observed in AML patients. Here, we report the development of an endogenously targeted, Flp inducible, Flt3 ITD mouse allele which can be somatically activated subsequent to cooperating disease alleles. When activated with a tamoxifen inducible FlpoER, Flt3 mutant mice developed rapid leukocytosis peaking at 4-6 weeks post activation and resolving by 8-10 weeks, a finding not previously observed in constitutive models. This leukocytosis was disproportionately monocytic and accompanied by pronounced anemia and thrombocytopenia. Long term, these mice develop a myeloproliferative disease , reminiscent of previously reported constitutive alleles. In competitive transplantation studies, Flt3 mutant cells initiated disease and outcompeted wild-type cells. Despite this competitive advantage, disease was incapable of transplanting into secondary recipients. We further observed a non-cell autonomous depletion of SLAM+ LSKs suggesting the Flt3 mutant cells cannot propagate disease in self-renewing stem cells. To evaluate how this allele influenced leukemic evolution we crossed this Flt3 ITD allele to a Flp inducible Npm1 c mouse where a pulse of tamoxifen simultaneously activated both alleles. The combination of mutant Npm1 and Flt3 resulted in progressive leukocytosis which did not resolve. Within 6 weeks of mutational activation, these mice developed a lethal AML with robust anemia, thrombocytopenia, leukocytosis and expanded cKIT+ blasts in the blood. RNA-sequencing and immunophenotyping by CyTOF revealed distinct patterns of differentiation, gene-expression and downstream signaling.In an effort to model sequential mutational acquisition, we crossed the Flp Flt3 ITD allele to a Cre-inducible Dnmt3a R878H. Cre mRNA was electroporated into lineage negative bone marrow cells to activate the Dnmt3a R878H allele and transplanted into lethally irradiated recipients. Four weeks post engraftment, Flt3 ITD was activated with a pulse of tamoxifen. In contrast to the Flt3-Npm1 model, we observed an increase and subsequent decrease in WBC similar to the kinetics observed in Flt3 ITD only mice. However, by 20 weeks we observed a robust and consistent increase in WBC accompanied by an emergence of cKIT+ cells in the blood. Histopathology indicated that >50% of mice expressing both alleles in sequence developed AML marked by increased blasts in the marrow, with moderate anemia and thrombocytopenia compared to the Flt3-Npm1 models. Critically, in contrast to Flt3 ITD only mice, acquisition of the Flt3 ITD in Npm1 or Dnmt3a mutant HPSCs induced fully transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. Collectively these results demonstrate that different co-occurring mutations are capable of transforming Flt3 ITD mutant cells, albeit with distinct latencies and mechanisms of cooperativity. In summary, our studies utilizing novel multi-recombinase models of leukemogenesis reveal new insights into the early phase of oncogene activation, and how cooperating alleles influence this response. This inducible Flt3 ITD allele represents a significant advance in modeling clonal evolution in myeloid malignancies and provides a critical isogenic platform for preclinical development of novel leukemia therapeutic regimens. Figure 1 Figure 1. Disclosures Bowman: Mission Bio: Honoraria, Speakers Bureau. Xiao: Stemline Therapeutics: Research Funding. Miles: Mission Bio: Honoraria, Speakers Bureau. Trowbridge: Fate Therapeutics: Patents & Royalties; H3 Biomedicine: Research Funding. Levine: Amgen: Honoraria; Lilly: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Ajax: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Incyte: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees.


2021 ◽  
Author(s):  
Robert B Jones ◽  
Jonathan Farhi ◽  
Milica Zecevic ◽  
Kiran Parwani ◽  
Miranda Adams ◽  
...  

PI3K pathway hyperactivation, including mutational activation of the PIK3CA isoform that encodes p110, is a common occurrence in hormone receptor-positive (HR+) breast cancer and has led to the clinical approval of the p110-selective inhibitor alpelisib. However, PI3K inhibition as a monotherapy lacks durability, at least in part due to aberrant downstream activation of histone H3 lysine 4 (H3K4) methylation. Here we show that inhibition of the H3K4 methyltransferase MLL1 in combination with PI3K inhibition reduces HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces AKT effector signaling and H3K4 methylation, MLL monotherapy may lead to PI3K effector hyperactivation, suggesting a feedback loop between MLL activity and AKT activation. We additionally show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4-directed methyltransferase MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and support the preclinical development and testing of pan-MLL inhibitors.


2021 ◽  
Author(s):  
Hyun Jae Cho ◽  
Rajeev Misra

Mutations are one of the common means by which bacteria acquire resistance to antibiotics. In an Escherichia coli mutant lacking major antibiotic efflux pumps AcrAB and AcrEF, mutations can activate alternative pathways that lead to increased antibiotic resistance. In this work, we isolated and characterized compensatory mutations of this nature mapping in four different regulatory genes—baeS, crp, hns, or rpoB. The gain-of-function mutations in baeS constitutively activated the BaeSR two-component regulatory system to increase the expression of the MdtABC efflux pump. The loss-of-function mutations in crp and hns caused de-repression of an operon coding for the MdtEF efflux pump. Interestingly, despite the dependence of rpoB missense mutations on MdtABC for their antibiotic resistance phenotype, neither the expression of the mdtABCDbaeSR operon nor that of other known antibiotic efflux pumps went up. Instead, the RNA-seq data revealed a gene expression profile resembling that of a “stringent” RNA polymerase where protein and DNA biosynthesis pathways were down-regulated, but pathways to combat various stresses were up-regulated. Some of these activated stress pathways are also controlled by the general stress sigma factor, RpoS. The data presented here also show that compensatory mutations can act synergistically to further increase antibiotic resistance to a level similar to the efflux pump-proficient parental strain. Together, the findings highlight a remarkable genetic ability of bacteria to circumvent antibiotic assault even in the absence of a major intrinsic antibiotic resistance mechanism. Importance Antibiotic resistance among bacterial pathogens is a chronic health concern. Bacteria possess or acquire various mechanisms of antibiotic resistance, and chief among them is the ability to accumulate beneficial mutations that often alter antibiotic targets. Here we explored E. coli’s ability to amass mutations in a background devoid of a major, constitutively expressed efflux pump and identified mutations in several regulatory genes that confer resistance by activating specific or pleiotropic mechanisms.


Blood ◽  
2021 ◽  
Author(s):  
Ana Patricia Silva ◽  
Afonso R.M. Almeida ◽  
Ana Cachucho ◽  
João L Neto ◽  
Sofie Demeyer ◽  
...  

Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of T-ALL patients display very high IL7R mRNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, on leukemogenesis remains unclear. Here, we show that overexpression of IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knock-in mice drives potential thymocyte self-renewal, and thymus hyperplasia due to increased proliferation of T-cell precursors, which subsequently infiltrate lymph nodes, spleen and bone marrow, ultimately leading to fatal leukemia. The tumors mimic key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of PI3K/Akt pathway that is paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing JAK/STAT, PI3K/Akt/mTOR and Notch signaling activation. Notably, we also find that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and can progress in the absence of IL-7, but remain sensitive to inhibitors of IL-7R-mediated signaling Ruxolitinib (Jak1), AZD1208 (Pim), Dactolisib (PI3K/mTOR), Palbociclib (Cdk4/6), and Venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that T-ALL patient samples with high wild type IL7R expression display a transcriptional signature resembling that from IL-7-stimulated pro-T cells and, critically, from IL7R mutant T-ALL cases. Overall, our studies demonstrate that high expression of IL-7Rα can promote T-cell tumorigenesis even in the absence of IL-7Rα mutational activation.


Author(s):  
Tatsunori Suzuki ◽  
Takahiro Kishikawa ◽  
Tatsuyuki Sato ◽  
Norihiko Takeda ◽  
Yuki Sugiura ◽  
...  

AbstractMutational activation of the KRAS gene occurs in almost all pancreatic ductal adenocarcinoma (PDAC) and is the earliest molecular event in their carcinogenesis. Evidence has accumulated of the metabolic reprogramming in PDAC, such as amino acid homeostasis and autophagic flux. However, the biological effects of KRAS mutation on metabolic reprogramming at the earlier stages of PDAC carcinogenesis are unclear. Here we report dynamic metabolic reprogramming in immortalized human non-cancerous pancreatic ductal epithelial cells, in which a KRAS mutation was induced by gene-editing, which may mimic early pancreatic carcinogenesis. Similar to the cases of PDAC, KRAS gene mutation increased the dependency on glucose and glutamine for maintaining the intracellular redox balance. In addition, the intracellular levels of amino acids were significantly decreased because of active protein synthesis, and the cells required greater autophagic flux to maintain their viability. The lysosomal inhibitor chloroquine significantly inhibited cell proliferation. Therefore, metabolic reprogramming is an early event in carcinogenesis initiated by KRAS gene mutation, suggesting a rationale for the development of nutritional interventions that suppress or delay the development of PDAC.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joshua H. Cook ◽  
Giorgio E. M. Melloni ◽  
Doga C. Gulhan ◽  
Peter J. Park ◽  
Kevin M. Haigis

AbstractMutational activation of KRAS promotes the initiation and progression of cancers, especially in the colorectum, pancreas, lung, and blood plasma, with varying prevalence of specific activating missense mutations. Although epidemiological studies connect specific alleles to clinical outcomes, the mechanisms underlying the distinct clinical characteristics of mutant KRAS alleles are unclear. Here, we analyze 13,492 samples from these four tumor types to examine allele- and tissue-specific genetic properties associated with oncogenic KRAS mutations. The prevalence of known mutagenic mechanisms partially explains the observed spectrum of KRAS activating mutations. However, there are substantial differences between the observed and predicted frequencies for many alleles, suggesting that biological selection underlies the tissue-specific frequencies of mutant alleles. Consistent with experimental studies that have identified distinct signaling properties associated with each mutant form of KRAS, our genetic analysis reveals that each KRAS allele is associated with a distinct tissue-specific comutation network. Moreover, we identify tissue-specific genetic dependencies associated with specific mutant KRAS alleles. Overall, this analysis demonstrates that the genetic interactions of oncogenic KRAS mutations are allele- and tissue-specific, underscoring the complexity that drives their clinical consequences.


2021 ◽  
Author(s):  
Qing Ye ◽  
Nasser Al-Kuwari ◽  
Pranay Srivast ◽  
Xiqun Chen

Abstract Background: Activating V600E in BRAF is a common driver mutation in cancers of multiple tissue origins, including melanoma and glioma. BRAF V600E has also been implicated in neurodegeneration. The present study aims to characterize BRAF V600E on cell death and survival in three major cell types of the CNS: neurons, astrocytes, and microglia. Methods : Multiple primary cultures and cell lines of glial cells and neurons were employed. BRAF V600E as well as BRAF WT expression was mediated by lentivirus or retrovirus. Blockage of downstream effectors were achieved by siRNA. Gene expression data from patients with Parkinson’s disease was analyzed. Results : In astrocytes and microglia, BRAF V600E induces cell proliferation, and the proliferative effect in microglia is mediated by activated ERK but not JNK. Conditioned medium from BRAF V600E -expressing microglia induced neuronal cell death. In neuronal cells, BRAF V600E directly induces cell death, through JNK but not ERK. We further show that BRAF-related genes are enriched in pathways in patients with Parkinson’s disease. Conclusions : Our study identifies distinct consequences mediated by distinct downstream effectors in dividing glial cells and in neurons following the same BRAF mutational activation and a causal link between BRAF-activated microglia and neuronal cell death that does not require physical proximity. It provides insight into a possibly important role of BRAF in neurodegeneration as a result of either dysregulated BRAF in neurons or its impact on glial cells.


2021 ◽  
Vol 2 ◽  
Author(s):  
Niels E. Wondergem ◽  
Dennis N. L. M. Nijenhuis ◽  
Jos B. Poell ◽  
C. René Leemans ◽  
Ruud H. Brakenhoff ◽  
...  

Background: Recent advances in immunotherapy for head and neck squamous cell carcinoma (HNSCC) have led to implementation of anti-programmed death receptor 1 (PD-1) immunotherapy to standard of care for recurrent/metastatic HNSCC. However, the majority of tumors do not respond to these therapies, indicating that these tumors are not immunogenic or other immunosuppressive mechanisms might be at play.Aim: Given their role in carcinogenesis as well as in immune modulation, we discuss the relation between the STAT3, PI3K/AKT/mTOR and Wnt signaling pathways to identify potential targets to empower the immune response against HNSCC.Results: We focused on three pathways. First, STAT3 is often overactivated in HNSCC and induces the secretion of immunosuppressive cytokines, thereby promoting recruitment of immune suppressive regulatory T cells and myeloid-derived suppressor cells to the tumor microenvironment (TME) while hampering the development of dendritic cells. Second, PI3K/AKT/mTOR mutational activation results in increased tumor proliferation but could also be important in HNSCC immune evasion due to the downregulation of components in the antigen-processing machinery. Third, canonical Wnt signaling is overactivated in >20% of HNSCC and could be an interesting pleotropic target since it is related to increased tumor cell proliferation and the development of an immunosuppressive HNSCC TME.Conclusion: The molecular pathology of HNSCC is complex and heterogeneous, varying between sites and disease etiology (i.e., HPV). The in HNSCC widely affected signaling pathways STAT3, PI3K/AKT/mTOR and Wnt are implicated in some of the very mechanisms underlying immune evasion of HNSCC, thereby representing promising targets to possibly facilitate immunotherapy response.


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