Holistic Review in Family Medicine Residency Programs: A CERA Study.

BACKGROUND AND OBJECTIVES: Interest in using holistic review for residency recruitment as a strategy to improve the diversity of the physician workforce has increased. However, no data are published on the prevalence of holistic review in the selection process for family medicine residency programs. We designed this study to assess programs' knowledge, skills, and attitudes; prevalence; barriers to implementation; and program characteristics associated with the use of holistic review. METHODS: Data for this study were elicited as part of a 2023 survey conducted by the Council of Academic Family Medicine Educational Research Alliance. The nationwide, web-based survey was sent to 739 family medicine residency program directors. RESULTS: A total of 309 program directors completed the holistic review portion of the survey. Programs that understood and agreed with holistic review used it more in their selection process. Holistic review was more common in programs with higher rates of residents, faculty, and patients that are underrepresented in medicine. Barriers to holistic review utilization were increased number of applicants, increased resources associated with holistic review, and lack of consensus on the holistic review approach. CONCLUSIONS: The holistic review process is an area of growing interest to diversify the physician workforce, especially among residencies caring for underresourced communities. Further discussions on the specific scoring rubrics of family medicine residency programs that use holistic review are needed and could help programs that are facing barriers. Widespread use of holistic review to diversify the physician workforce has the potential to improve patient care access and health.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

SHARPening Residency Selection: Implementing a Systematic Holistic Application Review Process.

PROBLEM: Traditional metrics used in residency application review processes are systematically biased against applicants from minoritized communities that are underrepresented in medicine (URiM). These biases harm not just URiM applicants but also residency programs and patients. Although several residency programs have implemented holistic reviews to mitigate these biases, few tested tools exist that can be adapted and implemented in a wide variety of settings within academic medicine. APPROACH: This article describes advances made in the third year of a longitudinal, ongoing quality improvement project that used the A3 framework to improve recruitment of URiM residents to a family medicine residency program. The authors devised a systematic holistic application review process (SHARP) to determine which applicants to invite to interview with the program. SHARP's development began in August 2019, and after significant discussion with program leadership and iterations of rubric refinement, the program adopted SHARP in September 2020 to review applications for the 2021 application cycle. OUTCOMES: Compared with the 2016 to 2020 period before SHARP implementation, data from the 2021 and 2022 residency application cycles after SHARP implementation showed a significant increase in the proportion of interviewed candidates who identify as URiM (from 23% to 38%, P < .001) and matched candidates who identify as URiM (from 27% to 62%, P = .004). There was also a notable increase in the number and diversity of reviewers who evaluated applicants to the program. NEXT STEPS: SHARP is a promising tool to mitigate the effects of racism and other biases against URiM applicants to residency programs. Residency programs across specialties may benefit from adopting SHARP and adapting it based on their own goals and priorities.

A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer.

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.

Glasses Versus Observation for Moderate Bilateral Astigmatism in 1- to <7-Year-Olds.

PURPOSE: To compare visual outcomes in children with moderate bilateral astigmatism treated with glasses with those who were merely observed. DESIGN: Retrospective case series. METHODS: The medical records of all children 1 to <7 years of age who were diagnosed with moderate bilateral astigmatism (+1.25 to +3.25 diopters [D]) at a single institution over a 12-year period were retrospectively reviewed. Children with anisometropia ≥1.00 D, hyperopia ≥+3.00 D, myopia ≥-3.00D, amblyopia, or strabismus at diagnosis were excluded. Observation or full spectacle correction of astigmatism was at the provider's discretion. Kaplan-Meier rates of developing amblyopia and strabismus were assessed over a minimum follow-up of 18 months. RESULTS: Eighty-five (6.9%) of 1235 subjects met the inclusion criteria; 58 (68.2%) were prescribed glasses while 27 (31.8%) were observed. The groups differed by mean age at diagnosis (3.56 ± 1.42 years for observed vs 4.31 ± 1.36 years for glasses [P = .03]) and mean amount of astigmatism (1.73 ± 0.43 D for observed vs 2.00 ± 0.51 D for glasses [P = .02]). By 4 years of follow-up, the Kaplan-Meier rate of developing amblyopia was 8.3% (95% confidence interval [CI] 0%-19.4%) in the observed group and 10.3% (95% CI 1.5%-19.1%) in the glasses group [P = .74] while strabismus was 7.1% (95% CI 0%-20.6%) among those observed and 7.1% (95% CI 0.4%-13.8%) of those prescribed glasses [P = .60]. CONCLUSIONS: Rates of amblyopia and strabismus were similar and modest in this cohort of children with moderate bilateral astigmatism treated with glasses vs observation. These results suggest that prescribing glasses for these children may be no better than observation in preventing the development of amblyopia or strabismus.

Visualization of endogenous p27 and Ki67 reveals the importance of a c-Myc-driven metabolic switch in promoting survival of quiescent cancer cells.

Rationale: Recurrent and metastatic cancers often undergo a period of dormancy, which is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the survival of quiescent cancer cells remain obscure. Methods: Dual genome-editing was carried out using a CRISPR/Cas9-based system to label endogenous p27 and Ki67 with the green and red fluorescent proteins EGFP and mCherry, respectively, in melanoma cells. Analysis of transcriptomes of isolated EGFP-p27highmCherry-Ki67low quiescent cells was conducted at bulk and single cell levels using RNA-sequencing. The extracellular acidification rate and oxygen consumption rate were measured to define metabolic phenotypes. SiRNA and inducible shRNA knockdown, chromatin immunoprecipitation and luciferase reporter assays were employed to elucidate mechanisms of the metabolic switch in quiescent cells. Results: Dual labelling of endogenous p27 and Ki67 with differentiable fluorescent probes allowed for visualization, isolation, and analysis of viable p27highKi67low quiescent cells. Paradoxically, the proto-oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27highKi67low quiescent cells and supported their survival through promoting mitochondrial oxidative phosphorylation (OXPHOS). In this context, c-Myc selectively transactivated genes encoding OXPHOS enzymes, including subunits of isocitric dehydrogenase 3 (IDH3), whereas its binding to cell cycle progression gene promoters was decreased in quiescent cells. Silencing of c-Myc or the catalytic subunit of IDH3, IDH3α, preferentially killed quiescent cells, recapitulating the effect of treatment with OXPHOS inhibitors. Conclusion: These results establish a rigorous experimental system for investigating cellular quiescence, uncover the high selectivity of c-Myc in activating OXPHOS genes in quiescent cells, and propose OXPHOS targeting as a potential therapeutic avenue to counter cancer cells in quiescence.

A systematic review of the sensitivity and specificity of lateral flow devices in the detection of SARS-CoV-2.

BACKGROUND: Lateral flow devices (LFDs) are viral antigen tests for the detection of SARS-CoV-2 that produce a rapid result, are inexpensive and easy to operate. They have been advocated for use by the World Health Organisation to help control outbreaks and break the chain of transmission of COVID-19 infections. There are now several studies assessing their accuracy but as yet no systematic review. Our aims were to assess the sensitivity and specificity of LFDs in a systematic review and summarise the sensitivity and specificity of these tests. METHODS: A targeted search of Pubmed and Medxriv, using PRISMA principles, was conducted identifying clinical studies assessing the sensitivity and specificity of LFDs as their primary outcome compared to reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2. Based on extracted data sensitivity and specificity was calculated for each study. Data was pooled based on manufacturer of LFD and split based on operator (self-swab or by trained professional) and sensitivity and specificity data were calculated. RESULTS: Twenty-four papers were identified involving over 26,000 test results. Sensitivity from individual studies ranged from 37.7% (95% CI 30.6-45.5) to 99.2% (95% CI 95.5-99.9) and specificity from 92.4% (95% CI 87.5-95.5) to 100.0% (95% CI 99.7-100.0). Operation of the test by a trained professional or by the test subject with self-swabbing produced comparable results. CONCLUSIONS: This systematic review identified that the performance of lateral flow devices is heterogeneous and dependent on the manufacturer. Some perform with high specificity but a great range of sensitivities were shown (38.32-99.19%). Test performance does not appear dependent on the operator. Potentially, LFDs could support the scaling up of mass testing to aid track and trace methodology and break the chain of transmission of COVID-19 with the additional benefit of providing individuals with the results in a much shorter time frame.

The Emerging Roles of RNA m6A Methylation and Demethylation as Critical Regulators of Tumorigenesis, Drug Sensitivity, and Resistance.

RNA N6 -methyladenosine (m6A) modification occurs in approximately 25% of mRNAs at the transcriptome-wide level. RNA m6A is regulated by the RNA m6A methyltransferases methyltransferase-like 3 (METTL3), METTL14, and METTL16 (writers), demethylases FTO and ALKBH5 (erasers), and binding proteins YTHDC1-2, YTHDF1-3, IGF2BP1-3, and SND1 (readers). These RNA m6A modification proteins are frequently upregulated or downregulated in human cancer tissues and are often associated with poor patient prognosis. By modulating pre-mRNA splicing, mRNA nuclear export, decay, stability, and translation of oncogenic and tumor suppressive transcripts, RNA m6A modification proteins regulate cancer cell proliferation, survival, migration, invasion, tumor initiation, progression, metastasis, and sensitivity to anticancer therapies. Importantly, small-molecule activators of METTL3, as well as inhibitors of METTL3, FTO, ALKBH5, and IGF2BP1 have recently been identified and have shown considerable anticancer effects when administered alone or in combination with other anticancer agents, both in vitro and in mouse models of human cancers. Future compound screening and design of more potent and selective RNA m6A modification protein inhibitors and activators are expected to provide novel anticancer agents, appropriate for clinical trials in patients with cancer tissues harboring aberrant RNA m6A modification protein expression or RNA m6A modification protein-induced resistance to cancer therapy.

The pan-cancer lncRNA PLANE regulates an alternative splicing program to promote cancer pathogenesis.

Genomic amplification of the distal portion of chromosome 3q, which encodes a number of oncogenic proteins, is one of the most frequent chromosomal abnormalities in malignancy. Here we functionally characterise a non-protein product of the 3q region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as E2F1-mediated transcriptional activation. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, thus leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. This is, at least in part, responsible for PLANE-mediated promotion of cancer cell proliferation and tumorigenicity. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

An Improved Protocol for Establishment of AML Patient-Derived Xenograft Models.

Patient-derived xenografts (PDXs) are the most valuable tool for preclinical drug testing because they retain the genetic diversity and phenotypic heterogeneity of the original tumor. Acute myeloid leukemia (AML) remains difficult to engraft in immunodeficient mice. This is particularly true for long-term frozen patient specimens. This protocol is designed to establish PDXs of human AML with improved engraftment rates. The optimized approach increases the viability of patient cells before implantation, efficiently monitors in vivo engraftment, and maximizes bone marrow collection. For complete details on the use and execution of this protocol, please refer to Salik et al. (2020) and Lynch et al. (2019).

Targeting RSPO3-LGR4 Signaling for Leukemia Stem Cell Eradication in Acute Myeloid Leukemia.

Signals driving aberrant self-renewal in the heterogeneous leukemia stem cell (LSC) pool determine aggressiveness of acute myeloid leukemia (AML). We report that a positive modulator of canonical WNT signaling pathway, RSPO-LGR4, upregulates key self-renewal genes and is essential for LSC self-renewal in a subset of AML. RSPO2/3 serve as stem cell growth factors to block differentiation and promote proliferation of primary AML patient blasts. RSPO receptor, LGR4, is epigenetically upregulated and works through cooperation with HOXA9, a poor prognostic predictor. Blocking the RSPO3-LGR4 interaction by clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts but does not affect normal hematopoietic stem cells, providing a therapeutic opportunity for HOXA9-dependent leukemia.

The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35.

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.

Diplopia after strabismus surgery for adults with nondiplopic childhood-onset strabismus.

PURPOSE: To describe frequency of postoperative diplopia after strabismus surgery in nondiplopic adults with childhood-onset strabismus and to report health-related quality-of-life (HRQOL) outcomes. METHODS: We prospectively enrolled 79 adults with no diplopia in any gaze who had childhood-onset strabismus and were scheduled for strabismus surgery. Diplopia was assessed preoperatively and at 6 weeks and 1 year postoperatively using a standardized diplopia questionnaire with 5 response options in 7 gaze positions. HRQOL was assessed using the Adult Strabismus-20 (AS-20) questionnaire, with self-perception, interactions, reading function, and general function domains. RESULTS: Constant diplopia in straight-ahead distance and reading gaze occurred in 1 patient (1% [95% CI, 0%-7%] at 6 weeks and 2% [95% CI, 0%-10%] at 1 year). Regarding the rate of any diplopia (including rarely) in any gaze, 15 of 78 patients (19%) reported diplopia at 6 weeks, of whom 13 had diplopia in straight-ahead distance gaze; 8 (10%), in reading gaze. At 1 year, 8 of 51 patients (16%) reported any diplopia (including rarely) in any gaze, of whom 7 had diplopia in straight-ahead distance gaze and 4 (8%) in reading gaze. Mean AS-20 scores improved at 1 year overall (by 32, 19, 14, and 15 points, resp., per domain) and for the 8 diplopic patients (by 21, 13, 16, and 11 points). CONCLUSIONS: In adults with nondiplopic strabismus, constant postoperative diplopia is rare, although the rate of intermittent diplopia is higher. Even when postoperative diplopia occurs, HRQOL often improves.

Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development.

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.

JMJD1C-mediated metabolic dysregulation contributes to HOXA9-dependent leukemogenesis.

Abnormal metabolism is a fundamental hallmark of cancer and represents a therapeutic opportunity, yet its regulation by oncogenes remains poorly understood. Here, we uncover that JMJD1C, a jumonji C (JmjC)-containing H3K9 demethylase, is a critical regulator of aberrant metabolic processes in homeobox A9 (HOXA9)-dependent acute myeloid leukemia (AML). JMJD1C overexpression increases in vivo cell proliferation and tumorigenicity through demethylase-independent upregulation of a glycolytic and oxidative program, which sustains leukemic cell bioenergetics and contributes to an aggressive AML phenotype in vivo. Targeting JMJD1C-mediated metabolism via pharmacologic inhibition of glycolysis and oxidative phosphorylation led to ATP depletion, induced necrosis/apoptosis and decreased tumor growth in vivo in leukemias co-expressing JMJD1C and HOXA9. The anti-metabolic therapy effectively diminished AML stem/progenitor cells and reduced tumor burden in a primary AML patient-derived xenograft. Our data establish a direct link between drug responses and endogenous expression of JMJD1C and HOXA9 in human AML cell line- and patient-derived xenografts. These findings demonstrate a previously unappreciated role for JMJD1C in counteracting adverse metabolic changes and retaining the metabolic integrity during tumorigenesis, which can be exploited therapeutically.