Search

Pediatric Epstein-Barr virus-negative monomorphic post solid organ transplant lymphoproliferative disorder [EBV(-)M-PTLD] comprises approximately 10% of M-PTLD. No large multi-institutional pediatric-specific reports on treatment and outcome are available.A multi-institutional retrospective review of solid organ recipients diagnosed with EBV(-)M-PTLD aged ≤21 years between 2001 and 2020 in 12 centers in the United States and United Kingdom was performed, including demographics, staging, treatment, and outcomes data.Thirty-six patients were identified with EBV(-)M-PTLD. Twenty-three (63.9%) were male. Median age (range) at transplantation, diagnosis of EBV(-)M-PTLD, and interval from transplant to PTLD were 2.2 years (0.1-17), 14 years (3.0-20), and 8.5 years (0.6-18.3), respectively. Kidney (n = 17 [47.2%]) and heart (n = 13 [36.1%]) were the most commonly transplanted organs. Most were Murphy stage III (n = 25 [69.4%]). Lactate dehydrogenase was elevated in 22/34 (64.7%) and ≥2 times upper limit of normal in 11/34 (32.4%). Pathological diagnoses included diffuse large B-cell lymphoma (n = 31 [86.1%]) and B-non-Hodgkin lymphoma (B-NHL) not otherwise specified (NOS) (n = 5 [13.9%]). Of nine different regimens used, the most common were: pediatric mature B-NHL-specific regimen (n = 13 [36.1%]) and low-dose cyclophosphamide, prednisone, and rituximab (n = 9 [25%]). Median follow-up from diagnosis was 3.0 years (0.3-11.0 years). Three-year event-free survival (EFS) and overall survival (OS) were 64.8% and 79.9%, respectively. Of the seven deaths, six were from progressive disease.EFS and OS were comparable to pediatric EBV(+) PTLD, but inferior to mature B-NHL in immunocompetent pediatric patients. The wide range of therapeutic regimens used directs our work toward developing an active multi-institutional registry to design prospective studies.Pediatric Epstein-Barr virus-negative monomorphic post solid organ transplant lymphoproliferative disorders (EBV(-)M-PTLD) have comparable outcomes to EBV(+) PTLD, but are inferior to diffuse large B-cell lymphoma in immunocompetent pediatric patients. The variety of treatment regimens used highlights the need to develop a pediatric PTLD registry to prospectively evaluate outcomes. The impact of treatment regimen on relapse risk could not be assessed because of small numbers. In the intensive pediatric B-non-Hodgkin lymphoma chemoimmunotherapy group, 11 of 13 patients remain alive in complete remission after 0.6 to 11 years.

The 1,287 genes targeted by the shRNA of the leukemia library.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

Hotspots difficult to confidently assess for CHIP

Plasmids.

Patient sample information.

Antibodies.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.Significance:Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.See related commentary by Li and Melnick, p. 198.

Supplementary Methods and Supplementary Figures

All supplemental figures and legends.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations usable for CHIP detection by performing a data mining analysis of 48 somatic mutation landscape studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with leukemia or other hematologic malignancy. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole-exome sequencing data from 4,538 persons belonging to three noncancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and precancer surveillance for both children and adults.Significance:This study identifies frequently occurring mutations across several blood cancers that may drive hematologic malignancies and signal increased risk for cancer when detected in healthy persons. We find clonal mutations at these hotspots in a substantial number of individuals from noncancer cohorts, including children, showcasing potential for improved precancer surveillance.See related commentary by Spitzer and Levine, p. 192.

Oligonucleotide sequences.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

Supplementary Figure 4

Supplementary Methods, Legends

Supplementary Figure 1

Supplementary Table 1

Supplementary Figure 3

Supplementary Figure 2

Purpose:Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis.Experimental Design:A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor).Results:JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo.Conclusions:Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Supplementary Table 2

Supplementary Table 3-8

Purpose Genetic laboratory test reports can often be of limited computational utility to the receiving clinical information systems, such as clinical decision support systems. Many health-care interoperability (HC) standards aim to tackle this problem, but the perceived benefits, challenges, and motivations for implementing HC interoperability standards from the labs' perspective has not been systematically assessed. Methods We surveyed genetic testing labs across the United States and conducted a semistructured interview with responding lab representatives. We conducted a thematic analysis of the interview transcripts to identify relevant themes. A panel of experts discussed and validated the identified themes. Results Nine labs participated in the interview, and 24 relevant themes were identified within five domains. These themes included the challenge of complex and changing genetic knowledge, the motivation of competitive advantage, provided financial incentives, and the benefit of supporting the learning health system. Conclusion Our study identified the labs' perspective on various aspects of implementing HC interoperability standards in producing and communicating genetic test reports. Interviewees frequently reported that increased adoption of HC standards may be motivated by competition and programs incentivizing and regulating the incorporation of interoperability standards for genetic test data, which could benefit quality control, research, and other areas.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Objective In many cases, genetic testing labs provide their test reports as portable document format files or scanned images, which limits the availability of the contained information to advanced informatics solutions, such as automated clinical decision support systems. One of the promising standards that aims to address this limitation is Health Level Seven International (HL7) Fast Healthcare Interoperability Resources Clinical Genomics Implementation Guide-Release 1 (FHIR CG IG STU1). This study aims to identify various data content of some genetic lab test reports and map them to FHIR CG IG specification to assess its coverage and to provide some suggestions for standard development and implementation. Materials and Methods We analyzed sample reports of 4 genetic tests and relevant professional reporting guidelines to identify their key data elements (KDEs) that were then mapped to FHIR CG IG. Results We identified 36 common KDEs among the analyzed genetic test reports, in addition to other unique KDEs for each genetic test. Relevant suggestions were made to guide the standard implementation and development. Discussion and Conclusion The FHIR CG IG covers the majority of the identified KDEs. However, we suggested some FHIR extensions that might better represent some KDEs. These extensions may be relevant to FHIR implementations or future FHIR updates. The FHIR CG IG is an excellent step toward the interoperability of genetic lab test reports. However, it is a work-in-progress that needs informative and continuous input from the clinical genetics’ community, specifically professional organizations, systems implementers, and genetic knowledgebase providers.

Simple Summary Myelofibrosis (MF) is a progressive myeloproliferative neoplasm with tendency towards leukemic transformation and has the poorest prognosis amongst the Philadelphia-negative classical myeloproliferative neoplasms (MPN). Ruxolitinib, the first FDA-approved JAK1/2 tyrosine kinase inhibitor, is efficient in reducing spleen size and improving patient symptoms, but it has not been shown to eradicate the MF clone. In this study, using functional genomics techniques, we identified the proteasome family as an additional therapeutic target and demonstrated that inhibition of the proteasome family by carfilzomib in combination with ruxolitinib enhanced suppression of primary MF cells in vitro. Abstract As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Purpose: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. Experimental Design: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). Results: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Conclusions: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations usable for CHIP detection by performing a data mining analysis of 48 somatic mutation landscape studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with leukemia or other hematologic malignancy. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole-exome sequencing data from 4,538 persons belonging to three noncancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and precancer surveillance for both children and adults. Significance: This study identifies frequently occurring mutations across several blood cancers that may drive hematologic malignancies and signal increased risk for cancer when detected in healthy persons. We find clonal mutations at these hotspots in a substantial number of individuals from noncancer cohorts, including children, showcasing potential for improved precancer surveillance. See related commentary by Spitzer and Levine, p. 192.

Genetic laboratory test reports can often be of limited computational utility to the receiving clinical information systems, such as clinical decision support systems. Many health-care interoperability (HC) standards aim to tackle this problem, but the perceived benefits, challenges, and motivations for implementing HC interoperability standards from the labs' perspective has not been systematically assessed.We surveyed genetic testing labs across the United States and conducted a semistructured interview with responding lab representatives. We conducted a thematic analysis of the interview transcripts to identify relevant themes. A panel of experts discussed and validated the identified themes.Nine labs participated in the interview, and 24 relevant themes were identified within five domains. These themes included the challenge of complex and changing genetic knowledge, the motivation of competitive advantage, provided financial incentives, and the benefit of supporting the learning health system.Our study identified the labs' perspective on various aspects of implementing HC interoperability standards in producing and communicating genetic test reports. Interviewees frequently reported that increased adoption of HC standards may be motivated by competition and programs incentivizing and regulating the incorporation of interoperability standards for genetic test data, which could benefit quality control, research, and other areas.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.

The availability of genetic test data within the electronic health record (EHR) is a pillar of the US vision for an interoperable health IT infrastructure and a learning health system. Although EHRs have been highly investigated, evaluation of the information systems used by the genetic labs has received less attention-but is necessary for achieving optimal interoperability. This study aimed to characterize how US genetic testing labs handle their information processing tasks.We followed a qualitative research method that included interviewing lab representatives and a panel discussion to characterize the information flow models.Ten labs participated in the study. We identified three generic lab system models and their relevant characteristics: a backbone system with additional specialized systems for interpreting genetic results, a brokering system that handles housekeeping and communication, and a single primary system for results interpretation and report generation.Labs have heterogeneous workflows and generally have a low adoption of standards when sending genetic test reports back to EHRs. Core interpretations are often delivered as free text, limiting their computational availability for clinical decision support tools. Increased provision of genetic test data in discrete and standard-based formats by labs will benefit individual and public health.

Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next-generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome-wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5∙39 × 10-5 , Bonferroni-adjusted), impacting leukaemia cell survival as the top-ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.

IMPORTANCE: Alterations in the IKZF1 gene drive B-cell acute lymphoblastic leukemia (B-ALL) but are not routinely used to stratify patients by risk because of inconsistent associations with outcomes. We describe a novel deletion in 22q11.22 that was consistently associated with very poor outcomes in patients with B-ALL with IKZF1 alterations. OBJECTIVE: To determine whether focal deletions within the λ variable chain region in chromosome 22q11.22 were associated with patients with B-ALL with IKZF1 alterations with the highest risk of relapse and/or death. DESIGN, SETTING, AND PARTICIPANTS: This cohort study included 1310 primarily high-risk pediatric patients with B-ALL who were taken from 6 independent clinical cohorts, consisting of 3 multicenter cohorts (AALL0232 [2004-2011], P9906 [2000-2003], and patients with Down syndrome who were pooled from national and international studies) and 3 single-institution cohorts (University of Utah [Salt Lake City], Children’s Hospital of Philadelphia [Philadelphia, Pennsylvania], and St. Jude Children’s Hospital [Memphis, Tennessee]). Data analysis began in 2011 using patients from the older studies first, and data analysis concluded in 2021. EXPOSURES: Focal 22q11.22 deletions. MAIN OUTCOMES AND MEASURES: Event-free and overall survival was investigated. The hypothesis that 22q11.22 deletions stratified the prognostic effect of IKZF1 alterations was formulated while investigating nearby deletions in VPREB1 in 2 initial cohorts (n = 270). Four additional cohorts were then obtained to further study this association (n = 1040). RESULTS: This study of 1310 patients with B-ALL (717 male [56.1%] and 562 female patients [43.9%]) found that focal 22q11.22 deletions are frequent (518 of 1310 [39.5%]) in B-ALL and inconsistent with physiologic V(D)J recombination. A total of 299 of 1310 patients with B-ALL had IKZF1 alterations. Among patients with IKZF1 alterations, more than half shared concomitant focal 22q11.22 deletions (159 of 299 [53.0%]). Patients with combined IKZF1 alterations and 22q11.22 deletions had worse outcomes compared with patients with IKZF1 alterations and wild-type 22q11.22 alleles in every cohort examined (combined cohorts: 5-year event-free survival rates, 43.3% vs 68.5%; hazard ratio [HR], 2.18; 95% CI, 1.54-3.07; P

Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB109.

Introduction. Family health history (FHx) is an important factor in breast and ovarian cancer risk assessment. As such, multiple risk prediction models rely strongly on FHx data when identifying a patient’s risk. These models were developed using verified information and when translated into a clinical setting assume that a patient’s FHx is accurate and complete. However, FHx information collected in a typical clinical setting is known to be imprecise and it is not well understood how this uncertainty may affect predictions in clinical settings. Methods. Using Monte Carlo simulations and existing measurements of uncertainty of self-reported FHx, we show how uncertainty in FHx information can alter risk classification when used in typical clinical settings. Results. We found that various models ranged from 52% to 64% for correct tier-level classification of pedigrees under a set of contrived uncertain conditions, but that significant misclassification are not negligible. Conclusions. Our work implies that (i) uncertainty quantification needs to be considered when transferring tools from a controlled research environment to a more uncertain environment (i.e, a health clinic) and (ii) better FHx collection methods are needed to reduce uncertainty in breast cancer risk prediction in clinical settings.

Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B's obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

Growth Factor Independence (GFI)1B coordinates assembly of transcriptional repressor complexes comprised of co-repressors and histone modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B’s obligate effector, Lysine Specific Demethylase (LSD)1. We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2 and ZNF217) in an LSD1-dependent manner to control erythroid fate specification. Among these, depletion of both HMG20A and HMG20B, or GSE1 block GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B—LSD1 binding. These findings demonstrate the central role of the GFI1B—LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.

Rapid conversion of chronic myeloid leukemia to chronic myelomonocytic leukemia in a patient on imatinib therapy

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾ 10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾ 2 ARCH genes and 52% had ⩾ 7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.

Objective Relationships were examined between persistent organic pollutants (POPs) and incident type 2 diabetes, end-stage renal disease (ESRD) and mortality. Methods In a nested case–control study, 300 persons without diabetes had baseline examinations between 1969 and 1974; 149 developed diabetes (cases) and 151 remained non-diabetic (controls) during 8.0 and 23.1 years of follow-up, respectively. POPs were measured at baseline. ORs for diabetes were computed by logistic regression analysis. The cases were followed from diabetes onset to ESRD, death or 2013. HRs for ESRD and mortality were computed by cause-specific hazard models. Patterns of association were explored using principal components analysis. Results PCB151 increased the odds for incident diabetes, whereas hexachlorobenzene (HCB) was protective after adjusting for age, sex, body mass index, sample storage characteristics, glucose and lipid levels. Associations between incident diabetes and polychlorinatedbiphenyl (PCB) or persistent pesticide (PST) components were mostly positive but non-significant. Among the cases, 29 developed ESRD and 48 died without ESRD. PCB28, PCB49 and PCB44 increased the risk of ESRD after adjusting for baseline demographic and clinical characteristics. Several PCBs and PSTs increased the risk of death without ESRD. The principal components analysis identified PCBs with low-chlorine load positively associated with ESRD and death without ESRD, and several PSTs associated with death without ESRD. Conclusions Most POPs were positively but not significantly associated with incident diabetes. PCB151 was significantly predictive and HCB was significantly protective for diabetes. Among participants with diabetes, low-chlorine PCBs increase the risk of ESRD and death without ESRD, whereas several PSTs predict death without ESRD.

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

AIMS/HYPOTHESIS: We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona. METHODS: Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA- DRA, and with type 2 diabetes mellitus and prediabetic traits. RESULTS: The A allele at rs9268852, which tags HLA-DRB1 *02 (1602), was associated both with higher HLA-DRB1 mRNA expression (n =133, p=4.27×10−(14)) and decreased risk of type 2 diabetes (n=3,265, OR 0.723, p=0.002). Among persons with normal glucose tolerance (n=266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p=0.005), higher mean 30 min insulin concentration during an oral glucose tolerance test (p=0.017) and higher body fat percentage (p=0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity. CONCLUSIONS/INTERPRETATION: HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.

Background Acute myeloid leukaemia (AML) is a tremendously heterogeneous clonal disorder of haemopoietic progenitor cells and is the most common malignant myeloid disorder in adults. Identified mutations from genomic data cannot provide information about their therapeutic significance without functional data. Hereby, we applied a pooled shRNA library screen to identify the activated signalling pathways essential for the survival of AML cells. Methods Mononuclear cells from seven karyotypically normal AML patients were separated from peripheral blood or bone marrow aspirate at diagnosis and transduced with a pooled shRNA library containing 27500 shRNAs targeting 5000 individual genes (Human Module 1, Decipher, Cellecta). The targeted genes were components of known signalling pathways. At 72h post transduction, 30% of the cells were stored for baseline measurements and the rest were co-cultured with HS-5 stromal cells for the selection period. DNA was then extracted and the shRNA barcodes were sequenced on the Illumina NextSeq platform. The frequency of barcode appearance after the selection period was compared to their prevalence at baseline to calculate the shRNA depletion. The shRNAs were filtered to identify those genes which had multiple shRNA meeting a threshold depletion. Results Our data analysis of the 7 AML samples identified various signalling pathways for each of these patients. These data support the notion of heterogeneity in AML. The top 100 depleted genes (with depletion in at least 3 shRNA per gene) in each patient were selected and compared. Our limited initial data showed there to be several activated signalling pathways for each AML sample indicating that inhibition of more than one gene or pathway might be required for efficiently suppressing these leukaemia cells. Common targets: NOX1 was the most commonly identified therapeutic target among the screened patients being significantly depleted in AML cells from 5/7 patients. This is an important finding as there are available NOX1 inhibitors for treatment of colon cancers and can be investigated as a therapeutic option for acute myeloid leukaemia. The other most common targets were CDK5R1, DISC1, FSCN3, and PSMB7 which were found to be significantly depleted among 3 of the 7 screened patients. The merged data also showed 58 essential genes for AML cell survival were common in at least 2/7 patients. Using Enrichr the activated signalling pathways based on the top selected genes were identified. Various signalling pathways were observed for each patient showcasing the heterogeneity among AML patients (Figure 1). However, some signalling pathways were indeed common among multiple patients - with different genes being responsible for the activation of those pathways among the patients. The most common pathway was the metabolic pathway which was observed among the top 20 essential pathways in 6/7 patients. The JAK-STAT5 signalling pathway, purine metabolism and cAMP signalling pathway were also among the top 20 essential pathways in 3/7 patients while the following pathways: FoxO, PI3K-AKT, HIF-1, P53, Glucagon, and proteasome were observed in 2/7 patients. Identification of several essential survival pathways provides the opportunity to develop personalised therapy through combined targeting of more than one pathway. Conclusion The signalling pathways analysis using candidate genes from a pooled shRNA library screen showed patient-specific signalling pathways and also common pathways among these screened patients. Absence of a common gene among the screened patients further highlights the significance of personalised therapy in AML and the necessity of developing diagnostic tools to identify potential targets at diagnosis. Identification of crucial genes such as NOX1 (a gene known to have a role in the survival of leukemic stem cells) and other genes with known significance in the pathogenesis of AML supports the application of this method for identifying therapeutic targets at diagnosis or relapse. Figure 1. Figure 1. Disclosures Knapper: Jazz: Other: Meeting and travel support; Daiichi Sankyo: Other: Meeting and travel support; Chroma Therapeutics: Research Funding; Celgene: Other: Meeting and travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Apperley:Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau.

OBJECTIVE We examined secular trends in the frequency distribution of albuminuria and estimated glomerular filtration rate (eGFR) in subjects with type 2 diabetes in 1982–1988 and 2001–2006, two periods associated with major changes in the management of diabetes. RESEARCH DESIGN AND METHODS The cross-sectional study included Pima Indians ≥15 years old with type 2 diabetes and measures of serum creatinine and urinary albumin-to-creatinine ratios (ACR). The continuous probability density distributions of ACR and eGFR were compared for the two time periods. eGFR was calculated using the Modification of Diet in Renal Disease Study equation. RESULTS The overall standardized distribution of ACR shifted toward lower values between time periods (P = 0.001), whereas the standardized distribution of eGFR did not (P = 0.45). In the first period, eGFR was CONCLUSIONS The distribution of albuminuria changed significantly among diabetic Pima Indians over the past 20 years, as treatment with medicines to control hyperglycemia and hypertension increased. The distribution of eGFR, however, remained unchanged. Consequently, the frequency of chronic kidney disease characterized by normoalbuminuria and low eGFR doubled.