Author Correction: Metabolic gatekeeper function of B-lymphoid transcription factors.

In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.

Metabolic gatekeeper function of B-lymphoid transcription factors.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

Genetic Counseling Assistants: an Integral Piece of the Evolving Genetic Counseling Service Delivery Model.

This study explores the potential impact of the genetic counseling assistant (GCA) position on the efficiency of the genetic counseling field, evaluates attitudes regarding expansion of the genetic counseling field to include the GCA, and presents data on GCA endeavors and GCA job tasks as reported by GCAs, certified genetic counselors (CGCs), and program directors (PDs). Data on GCA roles and attitudes toward different aspects of the GCA position were collected via surveys of CGCs who have worked with GCAs, PDs who have and have not had experience with GCAs in their programs, and GCAs. We analyzed responses from 63 individuals: 27 PDs, 22 CGCs, and 14 GCAs. GCAs' impact on efficiency was calculated via internal analysis of genetic patient volume per genetic counselor within the University of Texas Southwestern (UTSW) patient database prior to, and since the addition of, a GCA to the practice. The response rates for PDs, CGCs, and GCAs were 27 %, 79 %, and 61 %, respectively. Every CGC stated the GCA increased their efficiency. CGCs with a GCA reported a 60 % average increase in patient volume. This figure was congruent with internal data from the UTSW cancer genetics program (58.5 % increase). Appropriate responsibilities for GCAs as reported by CGCs and PDs (>90 %) include: data entry, shipping tests, administrative tasks, research, and ordering supplies. Regarding GCAs delivering test results, there was response variation whether this should be a job duty: 42 % of CGCs agreed to GCAs delivering negative results to patients, compared to 22 % of program directors. Twenty-two percent of PDs expressed concern about the job title "Genetic Counseling Assistant." Ninety percent of CGCs felt that GCA was a career path to becoming a CGC, compared to 42 % of PDs. Eighty-three percent of GCAs who decided to apply to CGC graduate programs were accepted. We conclude the addition of a GCA to a genetic counseling practice contributes to increased efficiency and is one way to expand the reach of the profession.

An internal performance assessment of CancerGene Connect: an electronic tool to streamline, measure and improve the genetic counseling process.

CancerGene Connect (CGC) is a web-based program that combines the collection of family and medical history, cancer risk assessment, psychosocial assessment, report templates, a result tracking system, and a patient follow up system. The performance of CGC was assessed in several ways: pre-appointment completion data analyzed for demographic and health variables; a time study to assess overall time per case and to compare the data entry by the genetic counselor compared to the patient, and a measured quality assessment of the program via observation and interview of patients. Prior to their appointment, 52.3% of 2,414 patients completed the online patient questionnaire section of CGC. There were significant differences in completion rates among racial and ethnic groups. County hospital patients were less likely to complete the questionnaire than insured patients (p < 0.0001); and likewise uninsured patients and patients with Medicare/Medicaid were less likely to complete the questionnaire than private patients (p < 0.0001). The average genetic counseling time per case was 82 min, with no significant differences whether the counselor or the patient completed CGC. CGC reduces genetic counselor time by approximately 14-46% compared to average time per case using traditional risk assessment and documentation methods previously reported. All surveyed users felt the questionnaire was easy to understand. CGC is an effective tool that streamlines workflow, and provides a standardized data collection tool that can be used to evaluate and improve the genetic counseling process.

Altered receptor trafficking in Huntingtin Interacting Protein 1-transformed cells.

The clathrin-associated protein, Huntingtin Interacting Protein 1 (HIP1), is overexpressed in multiple human epithelial tumors. Here, we report that HIP1 is a novel oncoprotein that transforms cells. HIP1-transformed cells, in contrast to RasV12-transformed cells, have dysregulation of multiple receptors involved in clathrin trafficking. Examples include upregulation of the epidermal growth factor receptor (EGFR) and the transferrin receptor. Furthermore, accumulation of transferrin and EGF in the HIP1-transformed cells was increased, and breast tumors that had EGFR expressed also had HIP1 upregulated. Thus, HIP1 overexpression promotes tumor formation and is associated with a general alteration in receptor trafficking. HIP1 is the first endocytic protein to be directly implicated in tumor formation.

BRCA1 and TP53 codeficiency causes a PARP inhibitor-sensitive erythroproliferative neoplasm.

Mutations in the BRCA1 tumor suppressor gene, such as 5382insC (BRCA1insC), give carriers an increased risk for breast, ovarian, prostate, and pancreatic cancers. We have previously reported that, in mice, Brca1 deficiency in the hematopoietic system leads to pancytopenia and, as a result, early lethality. We explored the cellular consequences of Brca1-null and BRCA1insC alleles in combination with Trp53 deficiency in the murine hematopoietic system. We found that Brca1 and Trp53 codeficiency led to a highly penetrant erythroproliferative disorder that is characterized by hepatosplenomegaly and by expanded megakaryocyte erythroid progenitor (MEP) and immature erythroid blast populations. The expanded erythroid progenitor populations in both BM and spleen had the capacity to transmit the disease into secondary mouse recipients, suggesting that Brca1 and Trp53 codeficiency provides a murine model of hematopoietic neoplasia. This Brca1/Trp53 model replicated Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib sensitivity seen in existing Brca1/Trp53 breast cancer models and had the benefits of monitoring disease progression and drug responses via peripheral blood analyses without sacrificing experimental animals. In addition, this erythroid neoplasia developed much faster than murine breast cancer, allowing for increased efficiency of future preclinical studies.

Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

Impact of a Cancer Gene Variant Reclassification Program Over a 20-Year Period.

PURPOSE: Hereditary cancer genetic testing can inform personalized medical management for individuals at increased cancer risk. However, many variants in cancer predisposition genes are individually rare, and traditional tools may be insufficient to evaluate pathogenicity. This analysis presents data on variant classification and reclassification over a 20-year period. PATIENTS AND METHODS: This is a retrospective analysis of > 1.9 million individuals who received hereditary cancer genetic testing from a single clinical laboratory (March 1997 to December 2017). Variant classification included review of evidence from traditional tools (eg, population frequency databases, literature) and laboratory-developed tools (eg, novel statistical methods, in-house RNA analysis) by a multidisciplinary expert committee. Variants may have been reclassified more than once and with more than one line of evidence. RESULTS: In this time period, 62,842 unique variants were observed across 25 cancer predisposition genes, and 2,976 variants were reclassified. Overall, 82.1% of reclassification events were downgrades (eg, variant of uncertain significance [VUS] to benign), and 17.9% were upgrades (eg, VUS to pathogenic). Among reclassified variants, 82.8% were initially classified as VUS, and 47.5% were identified in ≤ 20 individuals (allele frequency ≤ 0.001%). Laboratory-developed tools were used in 72.3% of variant reclassification events, which affected > 600,000 individuals. More than 1.3 million patients were identified as carrying a variant that was reclassified within this 20-year time period. CONCLUSION: The variant classification program used by the laboratory evaluated here enabled the reclassification of variants that were individually rare. Laboratory-developed tools were a key component of this program and were used in the majority of reclassifications. This demonstrates the importance of using robust and novel tools to reclassify rare variants to appropriately inform personalized medical management.

Huntingtin interacting protein 1 is a novel brain tumor marker that associates with epidermal growth factor receptor.

Huntingtin interacting protein 1 (HIP1) is a multidomain oncoprotein whose expression correlates with increased epidermal growth factor receptor (EGFR) levels in certain tumors. For example, HIP1-transformed fibroblasts and HIP1-positive breast cancers have elevated EGFR protein levels. The combined association of HIP1 with huntingtin, the protein that is mutated in Huntington's disease, and the known overexpression of EGFR in glial brain tumors prompted us to explore HIP1 expression in a group of patients with different types of brain cancer. We report here that HIP1 is overexpressed with high frequency in brain cancers and that this overexpression correlates with EGFR and platelet-derived growth factor beta receptor expression. Furthermore, serum samples from patients with brain cancer contained anti-HIP1 antibodies more frequently than age-matched brain cancer-free controls. Finally, we report that HIP1 physically associates with EGFR and that this association is independent of the lipid, clathrin, and actin interacting domains of HIP1. These findings suggest that HIP1 may up-regulate or maintain EGFR overexpression in primary brain tumors by directly interacting with the receptor. This novel HIP1-EGFR interaction may work with or independent of HIP1 modulation of EGFR degradation via clathrin-mediated membrane trafficking pathways. Further investigation of HIP1 function in brain cancer biology and validation of its use as a prognostic or predictive brain tumor marker are now warranted.

Aberrant Huntingtin interacting protein 1 in lymphoid malignancies.

Huntingtin interacting protein 1 (HIP1) is an inositol lipid, clathrin, and actin binding protein that is overexpressed in a variety of epithelial malignancies. Here, we report for the first time that HIP1 is elevated in non-Hodgkin's and Hodgkin's lymphomas and that patients with lymphoid malignancies frequently had anti-HIP1 antibodies in their serum. Moreover, p53-deficient mice with B-cell lymphomas were 13 times more likely to have anti-HIP1 antibodies in their serum than control mice. Furthermore, transgenic overexpression of HIP1 was associated with the development of lymphoid neoplasms. The HIP1 protein was induced by activation of the nuclear factor-kappaB pathway, which is frequently activated in lymphoid malignancies. These data identify HIP1 as a new marker of lymphoid malignancies that contributes to the transformation of lymphoid cells in vivo.

Functional Interaction of BRCA1 and CREBBP in Murine Hematopoiesis.

Both BRCA1 and CREBBP are tumor suppressor genes that are important for hematopoiesis. We have previously shown that mouse Brca1 is essential for hematopoietic stem cell (HSC) viability. In contrast to Brca1 deficiency, which results in pancytopenia, we report here that Crebbp deficiency results in myeloproliferation associated with an increase of splenic HSCs as well as a lethal systemic inflammatory disorder (LD50 = 86 days). To investigate the interaction of these two proteins in hematopoiesis, we generated double Crebbp/Brca1 knockout mice (DKOs). To our surprise, DKOs had accelerated bone marrow failure compared with Brca1-deficient mice and this was associated with an even shorter lifespan (LD50 = 88.5 versus 33 days). Furthermore, Crebbp or Brca1 heterozygosity influenced the hematopoietic phenotype associated with complete deficiency of Brca1 or Crebbp, respectively. We also observed lower BRCA1 protein levels in hematopoietic tissues when CREBBP is absent. Collectively, these data suggest Crebbp and Brca1 functionally interact to maintain normal hematopoiesis.

Family still matters: Counseling patients with complex family histories of colon and endometrial cancers.

BACKGROUND: There are no national guidelines for the management of patients with a family history consistent with Lynch syndrome (LS) but a negative genetic test. To determine current management practices, genetic counselors' (GCs) recommendations were assessed. METHODS: A survey of GCs using five hypothetical pedigrees was posted to National Society of Genetic Counselors (NSGC) discussion forums. Descriptive statistics were used. RESULTS: One-hundred and fifteen surveys were completed. A pedigree with a first-degree relative (FDR) with early-onset colorectal cancer (CRC) and a family history of CRC and endometrial cancer (EC) prompted 83% (n = 95) of respondents to recommend early and frequent colonoscopies, based on family history. When the CRCs and ECs occurred in family members removed from the proband, 96% (n = 110) of GCs said they would screen based on family history. However, only 52% (n = 60) suggested CRC screening should begin earlier and occur more often, and 43% (n = 50) suggested CRC screening should follow standard age and frequency guidelines. CONCLUSION: Concordance of opinion among GCs for the management of patients with negative genetic test results exists when FDRs are affected. However, when affected relatives are more distant, GCs disagreed on screening recommendations. These data suggest a need for guidelines for patients with a family history of cancer and a negative genetic test.

Deficiency of the Endocytic Protein Hip1 Leads to Decreased Gdpd3 Expression, Low Phosphocholine, and Kypholordosis.

Deficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated a Hip1 deficiency allele where a floxed transcriptional stop cassette and a human HIP1 cDNA were knocked into intron 1 of the mouse Hip1 locus. CMV-Cre-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of the Hip1 knockout phenotype. Mx1-Cre-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast, hGFAP-Cre-mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of several Hip1 knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC, Gdpd3 downregulation could lead to the low PC levels. To test whether Gdpd3 contributes to the Hip1 deficiency phenotype, we generated Gdpd3 knockout mice. Double knockout of Gdpd3 and Hip1 worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of how Hip1 deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.

Huntingtin-interacting protein 1 is overexpressed in prostate and colon cancer and is critical for cellular survival.

Huntingtin-interacting protein 1 (HIP1) is a cofactor in clathrin-mediated vesicle trafficking. It was first implicated in cancer biology as part of a chromosomal translocation in leukemia. Here we report that HIP1 is expressed in prostate and colon tumor cells, but not in corresponding benign epithelia. The relationship between HIP1 expression in primary prostate cancer and clinical outcomes was evaluated with tissue microarrays. HIP1 expression was significantly associated with prostate cancer progression and metastasis. Conversely, primary prostate cancers lacking HIP1 expression consistently showed no progression after radical prostatectomy. In addition, the expression of HIP1 was elevated in prostate tumors from the transgenic mouse model of prostate cancer (TRAMP). At the molecular level, expression of a dominant negative mutant of HIP1 led to caspase-9-dependent apoptosis, suggesting that HIP1 is a cellular survival factor. Thus, HIP1 may play a role in tumorigenesis by allowing the survival of precancerous or cancerous cells. HIP1 might accomplish this via regulation of clathrin-mediated trafficking, a fundamental cellular pathway that has not previously been associated with tumorigenesis. HIP1 represents a putative prognostic factor for prostate cancer and a potential therapy target in prostate as well as colon cancers.

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1.

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.

Serum antibodies to huntingtin interacting protein-1: a new blood test for prostate cancer.

Huntingtin-interacting protein 1 (HIP1) is frequently overexpressed in prostate cancer. HIP1 is a clathrin-binding protein involved in growth factor receptor trafficking that transforms fibroblasts by prolonging the half-life of growth factor receptors. In addition to human cancers, HIP1 is also overexpressed in prostate tumors from the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model. Here we provide evidence that HIP1 plays an important role in mouse tumor development, as tumor formation in the TRAMP mice was impaired in the Hip1null/null background. In addition, we report that autoantibodies to HIP1 developed in the sera of TRAMP mice with prostate cancer as well as in the sera from human prostate cancer patients. This led to the development of an anti-HIP1 serum test in humans that had a similar sensitivity and specificity to the anti-alpha-methylacyl CoA racemase (AMACR) and prostate-specific antigen tests for prostate cancer and when combined with the anti-AMACR test yielded a specificity of 97%. These data suggest that HIP1 plays a functional role in tumorigenesis and that a positive HIP1 autoantibody test may be an important serum marker of prostate cancer.

Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1F22-24/F22-24) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1BRCA1/BRCA1) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1F22-24/5382insC) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction.

HIP1: trafficking roles and regulation of tumorigenesis.

During recent years, alterations in proteins of the endocytic pathway have been associated with tumors. Disrupted regulation of the endocytic pathway is a relatively unstudied mechanism of tumorigenesis, which can concomitantly disrupt several different signaling pathways to affect growth, differentiation and survival. Several endocytic proteins have been identified, either as part of tumor-associated translocations or to have the ability to transform cells. Here, we summarize the information known about huntingtin interacting protein 1 (HIP1), an endocytic protein with transforming properties that is involved in a cancer-causing translocation and which is overexpressed in a variety of human cancers. We describe the known normal functions of HIP1 in endocytosis and receptor trafficking, the evidence for its role as an oncoprotein and how HIP1 might be altered to promote tumorigenesis.

Democratization of genetic data: connecting government approval of clinical tests with data sharing.

When a doctor orders a genetic test, patients assume that the test will yield a useful result to guide how their physicians take care of them. That assumption is frequently correct, but not always. Until recently, a genetic test only interrogated the sequence of one or two genes. Now, DNA-sequencing technologies are so fast and cheap that they have enabled clinicians to sequence panels of genes that may or may not be relevant to the patient's condition. The technology has outpaced our ability to interpret the results. Connecting approval of clinical tests to data sharing could help close this gap.