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.

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.

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.

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.

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.

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.

Use of Whole Genome Sequencing for Diagnosis and Discovery in the Cancer Genetics Clinic.

Despite the potential of whole-genome sequencing (WGS) to improve patient diagnosis and care, the empirical value of WGS in the cancer genetics clinic is unknown. We performed WGS on members of two cohorts of cancer genetics patients: those with BRCA1/2 mutations (n = 176) and those without (n = 82). Initial analysis of potentially pathogenic variants (PPVs, defined as nonsynonymous variants with allele frequency < 1% in ESP6500) in 163 clinically-relevant genes suggested that WGS will provide useful clinical results. This is despite the fact that a majority of PPVs were novel missense variants likely to be classified as variants of unknown significance (VUS). Furthermore, previously reported pathogenic missense variants did not always associate with their predicted diseases in our patients. This suggests that the clinical use of WGS will require large-scale efforts to consolidate WGS and patient data to improve accuracy of interpretation of rare variants. While loss-of-function (LoF) variants represented only a small fraction of PPVs, WGS identified additional cancer risk LoF PPVs in patients with known BRCA1/2 mutations and led to cancer risk diagnoses in 21% of non-BRCA cancer genetics patients after expanding our analysis to 3209 ClinVar genes. These data illustrate how WGS can be used to improve our ability to discover patients' cancer genetic risks.

Decreased BECN1 mRNA Expression in Human Breast Cancer is Associated with Estrogen Receptor-Negative Subtypes and Poor Prognosis.

Both BRCA1 and Beclin 1 (BECN1) are tumor suppressor genes, which are in close proximity on the human chromosome 17q21 breast cancer tumor susceptibility locus and are often concurrently deleted. However, their importance in sporadic human breast cancer is not known. To interrogate the effects of BECN1 and BRCA1 in breast cancer, we studied their mRNA expression patterns in breast cancer patients from two large datasets: The Cancer Genome Atlas (TCGA) (n=1067) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n=1992). In both datasets, low expression of BECN1 was more common in HER2-enriched and basal-like (mostly triple-negative) breast cancers compared to luminal A/B intrinsic tumor subtypes, and was also strongly associated with TP53 mutations and advanced tumor grade. In contrast, there was no significant association between low BRCA1 expression and HER2-enriched or basal-like subtypes, TP53 mutations or tumor grade. In addition, low expression of BECN1 (but not low BRCA1) was associated with poor prognosis, and BECN1 (but not BRCA1) expression was an independent predictor of survival. These findings suggest that decreased mRNA expression of the autophagy gene BECN1 may contribute to the pathogenesis and progression of HER2-enriched, basal-like, and TP53 mutant breast cancers.

Prediction of Cancer Prevention: From Mammogram Screening to Identification of BRCA1/2 Mutation Carriers in Underserved Populations.

BACKGROUND: The US Preventative Service Task Force recommends that physicians perform a genetic risk assessment to identify women at risk for BRCA1/2 mutations associated with hereditary breast and ovarian cancer (HBOC) syndrome. However, outcomes data after a diagnosis of HBOC syndrome especially in diverse populations, are minimal. Here we asked if genetic screening of high-risk underserved women identified in the mammogram population reduces cancer incidence. METHODS: We evaluated 61,924 underserved women at screening mammography for family histories suggestive of HBOC syndrome over the course of 21 months. Data were collected retrospectively from patients at two safety net hospitals through chart review. A computer model was used to calculate the long-term effect of this screening on cancer incidence by assessing both the mutation detection rate and the completion of prophylactic surgeries in BRCA1/2 mutation carriers. FINDINGS: We identified 20 of the 85 (23.5%) expected BRCA1/2 mutation carriers in the underserved population. The frequencies of prophylactic mastectomies and oophorectomies in the mutation carriers were 25% and 40%, respectively. Using these data, our model predicted only an 8.8% reduction in both breast and ovarian cancer in the underserved patients. This contrasts with a 57% reduction in breast cancer and 51% reduction in ovarian cancer in an insured reference population. Our data indicate that underserved patients with HBOC syndrome are difficult to identify and when identified are limited in their ability to adhere to NCCN guidelines for cancer prevention. INTERPRETATION: Screening for women at risk for HBOC syndrome in mammogram populations will only prevent cancers if we can increase compliance with management guidelines. This study provides prototypic baseline data for step-wise analysis of the efficacy of the use of family history analysis in the mammography setting for detection and management of HBOC syndrome.

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.

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.

Re-evaluating the role of BCR/ABL in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) requires the BCR/ABL tyrosine kinase for disease onset and maintenance. As a result, CML can be successfully treated with tyrosine kinase inhibitors (TKIs) such as imatinib. Most patients are maintained in a disease-suppressed state on daily TKI therapy for several years and in many cases this treatment prevents progression to the blast phase. If the TKI is discontinued, CML redevelops in 95% of patients as a result of persisting leukemia initiating cells (LICs). There are several hypotheses that describe the potential mechanism(s) responsible for LIC persistence in CML, but supporting evidence is limited. Furthermore, of the few patients who discontinue TKI therapy and are "cured" (i.e., in treatment-free remission), most have residual BCR/ABL-expressing cells in their hematopoietic tissues. There are also healthy individuals without a CML diagnosis who express the BCR/ABL mutation in a fraction of their hematopoietic cells. Finally, mice that express BCR/ABL from the Bcr locus as a knockin mutation do not develop CML. These mice have lower BCR/ABL levels than retroviral or transgenic models of BCR/ABL that do develop CML. Understanding why mice with BCR/ABL expressed from the Bcr locus and some people that express BCR/ABL are not afflicted with CML will provide insights into therapies to prevent or cure this disease.

Analysis of induced pluripotent stem cells from a BRCA1 mutant family.

Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.

Expression of BCR/ABL p210 from a knockin allele enhances bone marrow engraftment without inducing neoplasia.

Chronic myeloid leukemia (CML) and some acute lymphoblastic leukemias are characterized by the t(9;22) chromosome, which encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non-Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL-positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and nonleukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to control cells. These data suggest that BCR/ABL expression alone is insufficient to induce disease. This model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias.

Huntingtin-interacting protein 1 phosphorylation by receptor tyrosine kinases.

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor ß receptor (PDGFßR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFßR (H/P), and TEL/PDGFßR (T/P). We identified a four-tyrosine "HIP1 phosphorylation motif" (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFßR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival.