Resident-Reported Impact of a Novel Oncology Curriculum for Internal Medicine Residents.

The Accreditation Council of Graduate Medical Education mandates that all internal medicine residents gain exposure to internal medicine subspecialties including hematology and oncology. While many residents meet this criterion through inpatient oncology rotations, the current structure of many inpatient oncology rotations leaves little opportunity for formal education. We therefore designed a novel oncology curriculum consisting of one-page oncology teaching sheets to increase the number, breadth, and quality of formal teaching sessions on our resident inpatient oncology services. In order to evaluate the curriculum, we conducted pre- and post-intervention surveys of residents. From these surveys, we found that 72.2% of residents used the teaching sheets on their inpatient oncology rotation and that the teaching sheets led to an increase in the number of formal oncology teaching sessions (mean 3.4 ± 2.1 post-implementation vs 2.6 ± 2.0 pre-implementation, p = 0.008), the breadth of oncology topics taught (% reporting ≥ 5 topics; 26.1% vs 16.3%, p = 0.035), the proportion of residents reporting improvement in overall oncology knowledge (80.2% vs 62.4%, p = 0.012), and the proportion of residents reporting improvement in their ability to care for patients (70.8% vs 48.9%, p = 0.013). These results demonstrate that formal oncology teaching can be improved on inpatient oncology rotations through a simple and easily replicable oncology curriculum.

Inherited Platelet Disorders.

Bleeding disorders due to platelet dysfunction are a common hematologic complication affecting patients, and typically present with mucocutaneous bleeding or hemorrhage. An inherited platelet disorder should be suspected in individuals with a suggestive family history and no identified secondary causes of bleeding. Genetic defects have been described at all levels of platelet activation, including receptor binding, signaling, granule release, cytoskeletal remodeling, and platelet hematopoiesis. Management of these disorders is typically supportive, with an emphasis on awareness, patient education, and anticipatory guidance to prevent future episodes of bleeding.

The clinical and functional effects of TERT variants in myelodysplastic syndrome.

Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of a telomere biology disorder who underwent allogeneic transplantation. Patients with a TERT rare variant had shorter telomere length (P < .001) and younger age at MDS diagnosis (52 vs 59 years, P = .03) than patients without a TERT rare variant. In multivariable models, TERT rare variants were associated with inferior overall survival (P = .034) driven by an increased incidence of nonrelapse mortality (NRM; P = .015). Death from a noninfectious pulmonary cause was more frequent among patients with a TERT rare variant. Most variants were missense substitutions and classified as variants of unknown significance. Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 90% of TERT rare variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized. Routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion may identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.

Clonal hematopoiesis in the inherited bone marrow failure syndromes.

Inherited bone marrow failure syndromes (IBMFSs) are characterized by ineffective hematopoiesis and increased risk for developing myeloid malignancy. The pathophysiologies of different IBMFSs are variable and can relate to defects in diverse biological processes, including DNA damage repair (Fanconi anemia), telomere maintenance (dyskeratosis congenita), and ribosome biogenesis (Diamond-Blackfan anemia, Shwachman-Diamond syndrome). Somatic mutations leading to clonal hematopoiesis have been described in IBMFSs, but the distinct mechanisms by which mutations drive clonal advantage in each disease and their associations with leukemia risk are not well understood. Clinical observations and laboratory models of IBMFSs suggest that the germline deficiencies establish a qualitatively impaired functional state at baseline. In this context, somatic alterations can promote clonal hematopoiesis by improving the competitive fitness of specific hematopoietic stem cell clones. Some somatic alterations relieve baseline fitness constraints by normalizing the underlying germline deficit through direct reversion or indirect compensation, whereas others do so by subverting senescence or tumor-suppressor pathways. Clones with normalizing somatic mutations may have limited transformation potential that is due to retention of functionally intact fitness-sensing and tumor-suppressor pathways, whereas those with mutations that impair cellular elimination may have increased risk for malignant transformation that is due to subversion of tumor-suppressor pathways. Because clonal hematopoiesis is not deterministic of malignant transformation, rational surveillance strategies will depend on the ability to prospectively identify specific clones with increased leukemic potential. We describe a framework by which an understanding of the processes that promote clonal hematopoiesis in IBMFSs may inform clinical surveillance strategies.

Bridging the Divide: Student Grand Rounds at the Interface of Basic Science and Clinical Medicine.

PROBLEM: As biomedical research and clinical medicine become increasingly complex, physician-scientists and clinically oriented biomedical researchers play important roles in bridging the gap between disciplines. A lack of educational programming that addresses the unique needs of students preparing for careers at the interface of basic science and clinical medicine may contribute to trainee attrition. APPROACH: The MD-PhD/LHB Grand Rounds was introduced in 2008 as a trainee-driven collaborative effort of the Harvard/Massachusetts Institute of Technology MD-PhD program at Harvard Medical School (HMS MD-PhD program), Harvard's Leder Human Biology and Translational Medicine (LHB) program, and the Brigham and Women's Hospital (BWH) Internal Medicine Department. Each of the program's approximately 4 sessions per year begins with dinner, followed by a clinical case presentation led by a BWH MD-PhD resident with a master clinician faculty discussant, then a research presentation by an LHB PhD student or an MD-PhD student on a basic science topic related to the clinical case, and time for socialization. OUTCOMES: In a July 2017 survey of participating students and residents, respondents reported being highly satisfied with the program. Mean satisfaction ratings were 4.3 (SD 0.5) for 12 MD-PhD students, 4.2 (SD 0.7) for 31 LHB students, and 4.4 (SD 0.9) for 5 residents on a 5-point scale (5 = very satisfied). Free-text responses suggested MD-PhD students valued opportunities for active engagement with the resident presenter and faculty discussant. LHB students appreciated the absence of medical jargon in the clinical presentations. Residents' reported reasons for participating included enjoyment of teaching and interaction with students. NEXT STEPS: The Harvard MD-PhD/LHB Grand Rounds can serve as a template for developing similar programs at other institutions. Research is needed to determine whether such grand rounds programs can help fix the leaky pipeline in the training of future physician-scientists and clinically oriented biomedical researchers.

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif.

The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti-K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.

Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation.

Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl-exchange chemistry. As such, tritiated palmitate remains a readily accessible and direct method for monitoring the palmitoylation status of Ras proteins under a multitude of conditions.

Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho-K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL-dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP3 regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.

FKBP12 binds to acylated H-ras and promotes depalmitoylation.

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.

A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1.

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.