Activating Germline Mutations Research Articles

WHIM-синдром: обзор литературы и описание двух собственных клинических наблюдений в одной семье

WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis) is a rare genetic disease associated with activating germline mutations in the gene encoding chemokine receptor CXCR4. WHIM syndrome is manifested by neutropenia, lymphopenia, infections, and degenerative changes of mature neutrophils with bone marrow myeloid hyperplasia (myelokathexis). Some patients show hypogammaglobulinemia, persistent cutaneous, genital, or elsewhere localized warts. There are also cases of congenital heart defects. The present paper extensively analyzes genetic basis, pathophysiology, clinical manifestations, and diagnosis of WHIM syndrome as well as its treatment options. The paper reports two cases in one family.

Medullary thyroid cancer: molecular factors, management and treatment.

Medullary thyroid cancer (MTC) is an infrequent neuroendocrine tumor, which amounts to 3–5% of all thyroid malignancies. Approximately 75–80% of MTCs are sporadic neoplasms. The rest of 20–25% are familial cases that belong to multiple endocrine neoplasia (MEN) syndromes, specifically MEN2 and MEN3. These cases of familial MTC are attributed to an activating germline mutation of a tyrosine kinase receptor gene, the rearranged during transfection (RET) proto-oncogene, located on chromosome 10q11.21. These mutations are also found in some cases of sporadic MTC. This review sets forth in summary the accepted guidelines and approaches regarding diagnosis, management, and treatment of MTC. Surgical resection is the standard care, and an early, prophylactic intervention is performed in genetic cases. Further investigation and understanding of the molecular pathways involved in the growth and advancement of MTC is required in order to provide efficient therapy in cases of progressive disease.

Recombinant GH treatment in a case of Costello syndrome with a 5-year follow-up

.Costello syndrome (CS) is a rare member of the group of neuro-cardio-facio-cutaneous diseases known as RASopathies. CS involves characteristic dysmorphic craniofacial features, cardiac defects, and increased cancer susceptibility, depending on the heterozygous activating germline mutations in HRAS. Polyhydramnios and high birth weight are the most common presentations in the perinatal and neonatal periods; while poor postnatal growth, short stature, and failure to thrive are significant issues in infancy. Possible mechanisms of short stature in CS include GH deficiency and feeding difficulties. Only a few reported cases of CS with GH deficiency exist in literature. Here, we describe the 5-yr follow-up of a CS patient with complete GH deficiency treated with recombinant human GH (rhGH) from the age of four years. No significant adverse events regarding progression of hypertrophic cardiomyopathy and tumor development were observed. She has been responsive to treatment with improved growth velocity and height standard deviation scores. She is still under continuous monitoring for concerns on the possible development of cardiac events and malignancies. This case indicated that rhGH therapy is effective for improving the height and growth velocity of CS patients with GH deficiency under close cardiac and oncological monitoring.

Case Reports of Familial Non-autoimmune Hyperthyroidism in Two Malaysian Families due to Germline-activating Mutations in the Thyrotropin Receptor Gene

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Targeting Unique Synthetic Lethal Interactions between PI3K and MYC in B-ALL

Significance: Activating mutations of PIK3CA and other PI3K subunits are frequent in almost all types of cancer. Likewise, inactivating lesions of PTEN are very common in almost all cancer types. However, activating of PI3KCA and deleterious mutations of PTEN were not found in any of the 987 cases of B-ALL in our study. These findings are in line with recent studies suggesting that rare activating germline mutations in the PI3K pathway predispose to cancer but generally cause B-lymphopenia. Here we studied the underlying reasons for the remarkable sensitivity of normal B-cell precursors and B-ALL and examined strategies to leverage PI3K hyperactivation for therapeutic purposes. Results: Our study revealed the dysregulated glucose metabolism in PTEN-deficient pre-B ALL cells, which is indicated by increased glucose consumption, lactate production and deprived cellular ATP levels. After closely examining gene expression profile of PTEN KO murine ALL cells, we found glutaminolysis related enzymes and transporters were broadly decreased after PTEN deletion. Considering the observed ATP deficit and decreased glutaminolysis genes expression in PTEN KO ALL cells, we further performed Seahorse analysis and glutamine consumption assay. The results showed significant half-fold reduction of both oxygen consumption rate and glutamine utilization in PTEN KO ALL cells. We noticed most of the downregulated glutaminolysis genes are reported MYC targets and observed significantly decreased Myc protein levels both in PTEN KO and Pik3caH1047 overexpressed murine ALL cells. Furthermore, abrogation of PTEN activity by selective PTEN inhibitor SF1670 decreased MYC protein levels in patient-derived pre-B ALL xenografts. Those data indicate MYC as a metabolic activator is negatively regulated by PI3K signaling in pre-B ALL cells. Considering the hyperactivation of PI3K signaling after PTEN deletion, we performed CRISPR-RNP mediated genetic deletion of key PI3K-adaptor protein genes to compromise PI3K signaling in ALL cells and hopefully mitigate their sensitivity to PTEN deletion. Nevertheless, deletion of one or two of those genes including Cd19, Pik3ap1 and Irs1 did not alleviate PTEN-deletion caused cell death even with slightly decreased basal PI3K signaling level. Based on the observation of reduced levels of MYC protein and its target genes, we assessed the effect of Myc overexpression in rescuing PTEN-deletion caused cell death. Growth competition assay as well as cell-viability analysis showed profound rescue effects of Myc overexpression in PTEN KO ALL cells. More importantly, Myc overexpression not only increased cellular respiration and glutamine consumption but also recovered ATP levels in those cells. Meanwhile, we found even the overexpressed Myc protein level was reduced upon PTEN deletion, which indicates decreased Myc protein stability after PI3K hyperactivation. By overexpressing the MycT58A mutant, we found that proteasomal degradation resistant Myc mutant showed 2-fold enhanced rescue effect than wild type Myc in PTEN KO ALL cells. Our results demonstrate a PI3K-mediated degradation of MYC and its role in regulating glucose and glutamine metabolism in pre-B ALL. Conclusions: Unlike other cell types, B-cells are under particular metabolic constraints owing to repression of glucose uptake by B-lymphoid transcription factors PAX5 and IKZF1 (Chan et al., Nature 2017; Xiao et al., Cell 2018). As a result of the unique metabolic gatekeeper activity of B-cell-specific PAX5 and IKZF1-function, B-cells survive under conditions of chronic energy deprivation, which makes them particularly susceptible to acute fluctuation of energy expenditure, e.g. by activation of PI3K upon deletion of PTEN. In addition to increasing energy demands by acute PI3K-activation, PTEN-deletion also results in accelerated degradation of MYC, which could otherwise replenish glucose and energy levels. Strikingly, ectopic expression of ubiquitination-resistant T58A-Myc dramatically rescued cell death, which reveals a previously unrecognized synthetic lethality in B-ALL cells. Based on these observation, we propose dual targeting of PTEN with the small molecule phosphatase inhibitor SF1670 (Shojaee et al., Nat Med 2016) with inhibition of glutaminolysis, which erases the rescue effect of Myc on metabolic depletion. Disclosures No relevant conflicts of interest to declare.

Costello syndrome: Clinical phenotype, genotype, and management guidelines.

Costello syndrome (CS) is a RASopathy caused by activating germline mutations in HRAS. Due to ubiquitous HRAS gene expression, CS affects multiple organ systems and individuals are predisposed to cancer. Individuals with CS may have distinctive craniofacial features, cardiac anomalies, growth and developmental delays, as well as dermatological, orthopedic, ocular, and neurological issues; however, considerable overlap with other RASopathies exists. Medical evaluation requires an understanding of the multifaceted phenotype. Subspecialists may have limited experience in caring for these individuals because of the rarity of CS. Furthermore, the phenotypic presentation may vary with the underlying genotype. These guidelines were developed by an interdisciplinary team of experts in order to encourage timely health care practices and provide medical management guidelines for the primary and specialty care provider, as well as for the families and affected individuals across their lifespan. These guidelines are based on expert opinion and do not represent evidence-based guidelines due to the lack of data for this rare condition.

MON-264 Familial Neonatal Nonautoimmune Hyperthyroidism Due To A Thyrotropin Receptor Gene Mutation (A619G).

Activating germline mutations of the TSH receptor are responsible for a rare form of non-autoimmune hyperthyroidism transmitted as an autosomal dominant trait. We describe the case of a patient and her mother presenting with neonatal non-autoimmune hyperthyroidism associated with a heterozygous A619G mutation previously described in one patient who presented in adolescence. Our patient is a 6 year old African-American female diagnosed with hyperthyroidism at 2 weeks of age. Thyroid tests were being followed in the NICU due to maternal history of hyperthyroidism. The mother had a history of hyperthyroidism and had thyroidectomy at age 4 years. The patient was thought to have neonatal Graves disease due to maternal history of hyperthyroidism. She was initially started on Propranolol at an outside hospital. She presented to the Children’s Hospital of Michigan Emergency Department at 7 weeks of age with tachycardia and constant crying. Her thyroid tests at that time revealed elevated free thyroxine and free T3 levels at 2.7 ng/mL (0.8-1.8) and 8.8 pg/mL (1.4- 4.4) respectively. TSH level was less than 0.008 µIU/mL (0.800-4.400). She had diffuse goiter. She was started on Methimazole and the dose was adjusted to keep her FT4 and T3 within normal range; TSH remained suppressed despite normalizing FT4 and T3 levels. Thyroid antibodies were repeatedly negative including Thyroid peroxidase, thyroid microsomal, thyroglobulin antibodies, thyrotropin receptor antibodies (TRab) and thyroid stimulating immunoglobulins (TSI). The patient also had bilateral exotropia and required strabismus surgery at 13 and at 17 months of age. She also has global developmental delay requiring physical, occupational, and speech therapy. Chromosomal microarray and fragile X DNA testing were normal. She is currently growing at 1.7 SDS for height and 0.8 SDS for weight and is on Methimazole 7.5 mg orally daily. TSHR sequencing and deletion/duplication analysis revealed that she is heterozygous for a missense variant: c.1856A>G; p.Asp619Gly. We report a case of a patient and her mother with non-autoimmune hyperthyroidism with a heterozygous c.1856A>G (p.Asp619Gly) missense variant in the TSHR gene. This variant has been previously identified in one individual in the compound heterozygous state and was considered to be the primary contributing mutation. Functional studies suggest that the variant leads to constitutive activity of the TSH receptor, supporting the pathogenicity of the variant. In our patient, the mutation was found to be inherited from the mother who also has a history of goiter and hyperthyroidism and unlike the previously reported case, our patient presented in the neonatal period. We describe the clinical course and associated findings in this family and discuss the evaluation and management of this interesting condition.

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a Bacillus anthracis lethal toxin (LeTx)-sensitive Nlrp1b allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1β and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1β and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive Nlrp1b allele.

Germline-activating mutations in PIK3CD compromise B cell development and function.

Gain-of-function (GOF) mutations in PIK3CD, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Abstract 5646: Germline activating mutations in the proto-oncogene GCM2 in primary hyperparathyroidism

Abstract Primary hyperparathyroidism (PHPT) is a common endocrine disease caused by parathyroid tumors (adenoma or hyperplasia). The hypersecreting parathyroid tumor elevates serum parathyroid hormone level, which causes hypercalcemia and the classical symptoms of PHPT, including kidney stones, osteoporosis, and neuromuscular symptoms associated with hypercalcemia, such as muscle weakness, drowsiness, and depression. Familial PHPT occurs in an isolated nonsyndromal form, termed familial isolated hyperparathyroidism (FIHP), or as part of a syndrome, such as multiple endocrine neoplasia type 1 or hyperparathyroidism-jaw tumor syndrome. We performed exome sequencing on germline DNA of eight index cases from eight unrelated kindreds with FIHP. Rare variants were identified (minor allele frequency &amp;lt;1%), and selected variants were assessed for co-segregation in affected family members by Sanger sequencing. Candidate genes were then screened in an additional 32 kindreds with FIHP. The functional consequence of variants in one FIHP candidate gene (GCM2, a parathyroid tissue-specific transcription factor) was analyzed in a cell culture model. In eight kindreds with FIHP, we identified three rare GCM2 missense variants. Functional characterization of GCM2 variants and deletion-analyses revealed a small C-terminal Conserved Inhibitory Domain (CCID). Two of the three rare variants were recurrent, located in the CCID that enhanced the transcriptional activity of GCM2, and found in seven of 40 (18%) kindreds with FIHP. Functional assays for transcriptional activity demonstrated that these two rare variants acted as gain-of-function mutations, suggesting that GCM2 is a parathyroid proto-oncogene. We further investigated the ethnicity of individuals with GCM2 CCID activating variants in PHPT patient samples and in genome survey datasets. Our results demonstrate that germline activating mutations in the GCM2 CCID can cause FIHP, and specific activating mutations expose a large population at risk for PHPT. Our findings of the first gene with mutations specific for FIHP are expected to have an important impact on clinical practice, including treatment decisions and genetic testing of individuals with PHPT and family members. Citation Format: Bin Guan, James M. Welch, Julie C. Sapp, Hua Ling, Meghana Vemulapalli, Yulong Li, Jennifer J. Johnston, Electron Kebebew, Leslie G. Biesecker, William F. Simonds, Stephen J. Marx, Sunita K. Agarwal. Germline activating mutations in the proto-oncogene GCM2 in primary hyperparathyroidism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5646. doi:10.1158/1538-7445.AM2017-5646

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation

Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.

GCM2-Activating Mutations in Familial Isolated Hyperparathyroidism

Primary hyperparathyroidism (PHPT) is a common endocrine disease characterized by parathyroid hormone excess and hypercalcemia and caused by hypersecreting parathyroid glands. Familial PHPT occurs in an isolated nonsyndromal form, termed familial isolated hyperparathyroidism (FIHP), or as part of a syndrome, such as multiple endocrine neoplasia type 1 or hyperparathyroidism-jaw tumor syndrome. The specific genetic or other cause(s) of FIHP are unknown. We performed exome sequencing on germline DNA of eight index-case individuals from eight unrelated kindreds with FIHP. Selected rare variants were assessed for co-segregation in affected family members and screened for in an additional 32 kindreds with FIHP. In eight kindreds with FIHP, we identified three rare missense variants in GCM2, a gene encoding a transcription factor required for parathyroid development. Functional characterization of the GCM2 variants and deletion analyses revealed a small C-terminal conserved inhibitory domain (CCID) in GCM2. Two of the three rare variants were recurrent, located in the GCM2 CCID, and found in seven of the 40 (18%) kindreds with FIHP. These two rare variants acted as gain-of-function mutations that increased the transcriptional activity of GCM2, suggesting that GCM2 is a parathyroid proto-oncogene. Our results demonstrate that germline-activating mutations affecting the CCID of GCM2 can cause FIHP.

A Nationwide Study of Multiple Endocrine Neoplasia Type 2A in Norway: Predictive and Prognostic Factors for the Clinical Course of Medullary Thyroid Carcinoma.

Multiple endocrine neoplasia type 2A (MEN 2A) is an autosomal dominant syndrome caused by activating germline mutations in the RET (REarranged during Transfection) proto-oncogene. MEN 2A has a strong (>95%) and age-dependent (5-25 years) clinical penetrance of medullary thyroid carcinoma (MTC). Several major studies have analyzed the predictive and prognostic factors for MEN 2A to find indicators that predict the optimal timing of prophylactic thyroidectomy. The aims of this study were to describe all known RET positive MEN 2A patients diagnosed in Norway and to evaluate the clinical course of MTC, as well as its predictive and prognostic factors. This nationwide retrospective cohort study included data for 65 (14 index and 51 screening patients) out of a total of 67 MEN 2A patients with the RET gene mutation who were diagnosed in Norway since 1974. Data were collected by reviewing patient files. The variables analyzed were genotype, phenotype, preoperative basal calcitonin, age at thyroid surgery, central lymph node dissection and nodal status at primary surgery, number of surgical procedures, and biochemical cure. Of the 65 patients, 60 had undergone thyroid surgery. The median follow-up period was 9.9 years. The patients were divided into pre-RET-and RET-era, which included patients who had thyroid surgery before January 1, 1994, and after, respectively. In index and screening patients, MTC was found, respectively, in 100% and 45% of cases, central lymph node dissection at primary surgery was done for 64% and 52% of patients, and the median total number of surgical procedures was two (range 1-6) and one (range 1-4). At primary surgery, all patients (n = 13) with lymph node metastases had preoperative basal calcitonin levels ≥68 pg/mL, and all patients (n = 17) without central lymph node dissection and preoperative basal calcitonin <40 pg/mL were biochemically cured. Multivariate analysis showed that preoperative basal calcitonin was a significant predictive factor for MTC superior to age at thyroid surgery when analyzing the entire period (p = 0.009) and the RET-era separately (p = 0.021). Prognostic factors for biochemical cure were preoperative basal calcitonin, central lymph node dissection, and nodal status at primary surgery (p = 0.037, p = 0.002, and p = 0.005) when analyzing the entire period, but only nodal status at primary surgery when the RET-era was considered separately (p = 0.006). Preoperative basal calcitonin alone can serve as an indicator for optimal timing and the extent of thyroid surgery for MEN 2A patients that could be considered safe. The results are consistent with previously reported data.

Abstract LB-133: Excessive activation of the Src-kinase HCK in the tumor stroma promotes progression of solid tumors

Abstract Hemopoietic cell kinase, HCK, is a Src-family kinase expressed in myeloid and B-lymphoid cells, regulates innate immune cell functions, and is aberrantly activated and/or expressed (incl HCK gene amplification) in solid tumors of the colon, stomach, lung and other organs. Here we exploit the Hck(CA) mouse model, which carries a constitutively activating germline mutation and spontaneously develops pulmonary inflammation, airspace enlargement and cellular consolidation highly reminiscent of patient with chronic obstructive pulmonary disease, COPD [1]. To establish whether aberrant activation of endogenous Hck promotes tumorigenesis, we tested three established cancer models. First, we found lung adenocarcinoma burden in Hck(CA)x Kras(LSL-G12D) mice compared to Kras(LSL-G12D) mice, where the oncogenic KrasG12D transgene becomes activated in response to transnasal administration of adenovirus-encoded Cre recombinase. Second, using gp130(F/+) mice as a strain predisposed to the development of intestinal-type gastric cancer [2], we established that compound Hck(CA)x gp130(F/+), developed tumor burden similar to that of gp130(F/F) animals. Furthermore, tumors from Hck(CA)x gp130(F/+) mice, but not from gp130(F/F) mice, showed sub-mucosal invasion. Third, we modeled sporadic colorectal cancer by weekly administration of the alkylating agent azoxymethane for 6 consecutive weeks [3]. Compared to wild-type mice, Hck(CA) mice exhibited increased tumor frequency and multiplicity, and colonic tumors in Hck(CA) mice showed mucosal invasion, which was absent from tumors in wild-type mice. In all models the Hck(CA) allele increased tumor cell proliferation and associated Stat3, Erk, Akt and S6 cytoplasmic signaling, although the total number of tumor-associated macrophages remained unchanged by the presence of the Hck(CA) allele. However gene expression profiling of the latter cells revealed excessive alternative macrophage activation from Hck(CA) mice with prominent expression of Il4, Il10, Il13, Arg1 and Ym1. Accordingly, adoptive transfer of Hck(CA) bone-marrow was sufficient to confer increase tumor burden and promote alternative macrophage polarisation, while reconstitution with triple Hck(KO)x Fgr(KO)x Lyn(KO) bone marrow reduced tumor burden. Collectively, our data suggest that mutations affecting the stroma, and macrophage polarization specifically, confer tumor progression and that HCK may provide a novel target for rational cancer drug development. References 1) J. Exp. Med. 196:589-604 (2002) 2) Nat Med 11:845 (2005) 3) Cancer Cell 24:25 (2013) Citation Format: Robert O'Donoghue, Ashleigh Poh, Adele Preaudet, Matthias Ernst. Excessive activation of the Src-kinase HCK in the tumor stroma promotes progression of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2015-LB-133

Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C

ObjectiveCongenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown....

Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.

Monogenic causes of autoimmunity give key insights to the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in 5 individuals with a spectrum of early-onset autoimmune disease including type 1 diabetes. These findings emphasise the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in Hyper IgE syndrome.

Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors

AbstractThe main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line–activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3–human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.

Breaking Bad: how does a neuronal protein cause neuroendocrine cancer?

Medullary thyroid carcinomas (MTC) are slow-growing tumors originating from neuroendocrine, parafollicular cells (C-cells) in the thyroid gland. Despite its low prevalence, MTC is one of the most deadly forms of thyroid cancers. Like other neuroendocrine cancers (e.g. pancreatic), MTC is often diagnosed at advanced stages at which surgical removal of the thyroid is no longer an effective treatment. MTC affects both men and women equally, is either sporadic (75% MTC cases) or hereditary (25% MTC cases) and associated to other neuroendocrine cancers (Multiple Endocrine Neoplasia syndrome, MEN2). About 20 years ago, a breakthrough discovery revealed that activating germline mutations in the Rearranged-during-transfection (RET) protooncogene are responsible for 100% hereditary forms of the disease and about 40% sporadic forms of MTC [1, 2]. As a result, a large effort was set out to understand how RET, which encodes a receptor tyrosine kinase, causes MTC. Furthermore, molecular therapies targeting RET and other receptors tyrosine kinase have been developed which have led to the recent approval of two new drugs by the FDA. However these drugs have limited benefits and a large proportion of MTC patients still have no access to an efficient treatment because the causes of sporadic MTC are not well understood [3]. In our study [4], we show that the protein kinase, cyclin-dependent kinase 5 (Cdk5), plays a crucial role in sporadic MTC proliferation and demonstrate that blocking Cdk5 activity or its downstream effectors reduces significantly MTC proliferation. Cdk5 and its activators are present in normal human thyroid tissues and are enriched in hereditary and sporadic MTC patient specimen. Furthermore specific targeting of Cdk5 activity or Cdk5 expression arrests the proliferation of sporadic MTC cell lines that were derived from patient tumors. We corroborated this result in vivo in a transgenic mouse line. Overexpression of the Cdk5 activator, p25, in mouse thyroid C cells leads to the development of MTC, while preventing p25 overexpression arrests tumor growth. These findings demonstrate clearly the importance of Cdk5 activity for MTC progression. This was at first intriguing because Cdk5 is better known as a neuronal protein. Indeed, Cdk5 and its activators are expressed at high levels in brain compared to other tissues. Cdk5 regulates brain development and function and deregulation of Cdk5 activity by binding to its cleaved activator, p25, has been linked to neurodegenerative diseases such as Alzheimer's disease. Considering that neuroendocrine cells and neurones have a common ontological origin (the neural crest) and that they share similar properties (e.g. calcium-dependent release of neurotransmitters or hormones), this study raises the possibility that common signalling pathways may underlie cancer and neurodegenerative diseases. Mechanistically we found that Cdk5 regulates the phosphorylation state of the tumor suppressor, retinoblastoma protein (Rb), and the subsequent expression of Cdk2 and cyclin A (Figure ​(Figure1A).1A). It is well accepted that cell cycle progression from G1 to S phase is dependent on the initial phosphorylation of Rb by cyclin D-Cdk4/6 and on the maintenance of Rb phosphorylated state by cyclinA/E-Cdk2 complexes [5]. Our findings suggest that this may not be true in neuroendocrine cells and that Cdk5 mediates the initial Rb phosphorylation instead of Cdk4/6. This may explain why certain cancer cell lines are resistant to Cdk4/6 inhibitors. To verify this hypothesis, it will be important to determine whether MTC cell lines are resistant to Cdk4/6 inhibitors and conversely whether Cdk4/6 resistant cells are sensitive to Cdk5 inhibitors. Figure 1 Cdk5 contributes to neuroendocrine thyroid cancer tumorigenesis In this study, we report a novel animal model for MTC based on a tetracycline-controlled transactivator system (tTA) in which we can induce or arrest MTC development by activating or repressing p25 overexpression in the mouse thyroid C cells (Figure ​(Figure1B).1B). We showed that Cdk5 activity was highly elevated in growing tumors compared to arrested tumors. Hence, we can generate and collect identical MTC tissues that differ only by their Cdk5 activation levels. By comparing the molecular content of arrested and growing tumors, we can gain a deep insight on the Cdk5-mediated mechanisms contributing to MTC progression. We used this comparative approach to identify Rb, Cdk2 and cyclin A as downstream effectors of Cdk5. Thus our animal model is a useful tool to identify new mechanisms underlying MTC tumorigenesis. In addition, this clinically relevant model of MTC can be used for the preclinical testing of new therapies. In conclusion, our study unravels a major role for Cdk5 in neuroendocrine thyroid cancer and suggests that targeting Cdk5 or downstream effectors is a valid strategy to cure MTC. Furthermore our animal model provides new routes to discover molecular pathways important for MTC and test potential therapies. Our findings represent an important advance in a field in which molecular tools are limited and the discovery of new putative drug targets difficult.