Search

Mutation of the essential splicing factor SF3B1 is primarily associated with hematological cancers but also occurs in solid tumors. We edited the most common mutation, K700E, into human embryonic stem (ES) cells to determine the effects of this mutation alone in an undifferentiated/non-cancer background. Unexpectedly, >20% of the significantly upregulated genes in the SF3B1K700E ES lines have immune functions. Thus, SF3B1 may have an additional role in proper expression of immune genes in appropriate cell types. In striking contrast, we found that published RNA-seq data from SF3B1 blood (MDS, CLL, AML) and non-blood (BRCA, UVM) cancers exhibited the opposite, downregulation of a multitude of immune pathways with 7 of the pathways shared among all 5 of the SF3B1 cancers. One of these pathways, “leukocyte migration”, is the 1st reported pathway shared among all splicing factor cancers, including the 5 SF3B1 cancers and MDS associated with U2AF1, SRSF2 and ZRSR2. Importantly, we identified CCR1, which is in the leukocyte migration pathway as the only shared downregulated gene in the 5 SF3B1 cancers and in U2AF1MT MDS. We conclude that downregulation of CCR1 and its associated immune pathway may play a key role in pathogenesis of these splicing factor cancers and are thus potential therapeutic targets.

Significance Terminal uridylyltransferases are writers of poly(U) tails in diverse RNA uridylation pathways. In the let-7 pathway, the LIN28:pre-let-7:TUTase ribonucleoprotein complex regulates the processing and maturation of the let-7 microRNA, and plays an important role in both physiology and disease. The presence of redundant TUTase orthologs and the diverse pathways poses challenges to revealing the molecular mechanisms of TUTases. We developed a precise tool to identify nanobodies that inhibited the LIN28:pre-let-7:TUTase interaction. The nanobody Nb-S2A4 specifically interacted with the LIN28:let-7 interaction fragment in TUT4, which is critical for oligouridylation and monouridylation of let-7. Our data illustrate that the nanobody tool is effective in distinguishing functions among protein orthologs and diverse pathways., The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3′ ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

Left ventricular aneurysms (LVA) are a somewhat infrequent condition that most often occur as the result of an acute myocardial infarction but also have other acquired and congenital etiologies. Preparing to care for these patients starts with a detailed preanesthetic evaluation that is not dissimilar to the evaluation required prior to other open cardiac procedures. However, there are unique points of focus including some specific imaging modalities. Additionally, the anesthetic management during the repair is similar to many other cardiac procedures requiring a bloodless field but with specialized hemodynamic considerations. This requires tailoring induction, monitoring and maintenance to both the pathophysiology of LVA and the patient’s unique comorbidities. While the average cardiac anesthesiologist should have a firm grasp of the involved monitoring and pharmacology, it is beneficial to review these concepts within the context of these factors. Furthermore, there are specific complications and pitfalls that require an understanding of both the procedure and disease process along with significant vigilance to successfully care for patients undergoing the SAVER/CABG procedure.

Patients suffering from valvular heart disease most commonly do so as the result of congenital abnormalities or acquired degenerative and infective processes. The impact of these disorders on quality of life is tremendous. Surgical correction represents the sole treatment for many of these conditions. The two most common procedures involving valvular heart disease are aortic valve and mitral valve surgery. This chapter reviews the anesthetic consideration for patients undergoing cardiac surgery for left and right heart valves.

High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.

Leukemia initiating cells (LICs) fuel leukemic growth and spark relapse. Previously thought to be primitive and rare, the LIC state may actually be heterogeneous and dynamic, enabling evasion of therapies. Here, we use single-cell transcriptomics to track LIC multipotency within the cellular ontogeny of MLL-rearranged B-lymphoblastic leukemia (MLL-r B-ALL). Although we identify rare transcriptionally and phenotypically primitive LICs, we also observe LICs emerging from more differentiated populations with the capability to replenish the full leukemic cellular diversity. We find that activation of MYC-driven oxidative phosphorylation controls this process of facultative state conversion in LICs.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.

SUMMARY LIN28 is an RNA-binding protein that regulates the maturation of the let-7 family of microRNAs by bipartite interactions with let-7 precursors through its two distinct cold shock and zinc-knuckle domains. Through inhibition of let-7 biogenesis, LIN28 functions as a pluripotency factor, as well as a driver of tumorigenesis. Here, we report a fluorescence polarization assay to identify small-molecule inhibitors for both domains of LIN28 involved in let-7 interactions. Of 101,017 compounds screened, six inhibit LIN28:let-7 binding and impair LIN28-mediated let-7 oligouridylation. Upon further characterization, we demonstrate that the LIN28 inhibitor TPEN destabilizes the zinc-knuckle domain of LIN28, while LI71 binds the cold shock domain to suppress LIN28’s activity against let-7 in leukemia cells and embryonic stem cells. Our results demonstrate selective pharmacologic inhibition of individual domains of LIN28 and provide a foundation for therapeutic inhibition of the let-7 biogenesis pathway in LIN28-driven diseases., In Brief LIN28 is an oncogenic protein that promotes transformation in malignancy by suppressing the maturation of let-7 microRNAs. Wang et al. developed a high-throughput screening strategy and identified inhibitors of LIN28 that restore mature let-7 levels., Graphical Abstract

Leukemia stem cells (LSCs) are the subset of leukemic cells that drive leukemia progression, resist therapy, and remain latent to spark disease relapse. LSCs are quantified by efficiency of xenografting immunodeficient mice, a measurement that is predictive of leukemia outcome. Although LSCs have been previously thought to be rare and phenotypically primitive, recent data indicate that LSCs may actually be heterogeneous. Here, we use single-cell transcriptomics combined with limiting dilution xenotransplantation to dissect the ontogeny of MLL-rearranged B-lymphoblastic leukemia (MLL-r B-ALL), an aggressive form of childhood leukemia. Compared to acute myeloid leukemia (AML), LSCs are abundant in MLL-r B-ALL. Recapitulating the unique clinical behavior of this form of leukemia, MLL-r B-ALL cells undergo a B-lymphoid to myeloid lineage switch under chemotherapy pressure consistent with primitive, multipotent transcriptional programs present in LSCs. Although we identify rare, chemotherapy-resistant, primitive LSCs, we also observe LSCs emerging from more differentiated populations. These facultative LSCs self-renew and possess the capability to replenish the full cellular diversity of MLL-r B-ALL. Using CITE-seq, we find that stem cell programs can be fully uncoupled from immunophenotype in MLL-r B-ALL. In mechanistic studies, we find that the phenotypically differentiated LSCs that drive bottom-up reconstitution of the leukemic cellular ontogeny bear signatures of MYC activation and oxidative phosphorylation. We confirm recruitment of these pathways in actively reconstituting, phenotypically differentiated LSCs, and define a pathway by which MYC rewires metabolism in MLL-r B-ALL LSCs. We find that MYC is required for LSC plasticity in vitro and in vivo. Targeting oxidative metabolism impairs LSC engraftment, identifying a potential therapeutic intervention. We conclude that the high LSC content and dual lineage and LSC plasticities of MLL-r B-ALL contribute to its chemotherapy resistance and persistently poor outcomes. Disclosures Shalek: Honeycomb Biotechnologies: Consultancy, Current equity holder in publicly-traded company; Cellarity: Consultancy, Current equity holder in publicly-traded company; Repertoire Immune Medicines: Consultancy, Current equity holder in publicly-traded company; Merck: Consultancy; Orche Bio: Consultancy, Current equity holder in publicly-traded company; Dahlia Biosciences: Consultancy, Current equity holder in publicly-traded company.

With age, HSCs accumulate somatic mutations that can increase their relative fitness and cause clonal expansion, triggering sporadic age-related clonal hematopoiesis (CH). In most people, CH is of little or no clinical significance as mature blood cell output is not typically affected. However, some individuals will accumulate further mutations that drive evolution to HSC failure, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Moreover, in patients with bone marrow failure syndromes caused by germline mutations that impair HSC function, this process of CH is markedly accelerated, with an amplified risk of MDS and AML in young adulthood. Here, we aimed to use gene editing and directed differentiation of human induced pluripotent stem cells (IPSCs) to model sporadic and predisposed CH. We found that hematopoietic stem and progenitor cells (HSPCs) derived from IPSCs and deficient for the Fanconi anemia gene FANCA showed poor hematopoietic potential compared to isogenic FANCA-expressing cells. Multiplexed editing of 10 CH-associated genes in FANCA-deficient HSPCs resulted in reversal of this hematopoietic failure and implementation of an aberrant program of sustained self-renewal. To model sporadic CH, we introduced a heterozygous P95H mutation in the SRSF2 gene - a mutation commonly observed in human CH and MDS. We found that in HSPCs, this mutation globally disrupted splicing, mildly impaired differentiation, and enhanced clonogenesis. Together, our data demonstrate that we can capture hallmarks of predisposed and sporadic CH in IPSC-based models as a basis for ongoing studies on mechanisms of clonal evolution in HSCs.

Leukemia initiating cells (LICs) are the subset of leukemic cells that drive leukemia progression, resist therapy, and spark disease relapse. Although LICs have been previously thought to be rare and phenotypically primitive, recent data suggest that the LIC state may actually be heterogeneous. Here, we use single-cell transcriptomics combined with limiting dilution xenotransplantation to dissect the ontogeny of MLL-rearranged B-lymphoblastic leukemia (MLL-r B-ALL), an aggressive form of childhood leukemia. Compared to acute myeloid leukemia (AML), LICs are abundant in MLL-r B-ALL. Recapitulating the unique clinical behavior of this form of leukemia, MLL-r B-ALL cells undergo a B-lymphoid to myeloid lineage switch under chemotherapy pressure consistent with primitive, multipotent transcriptional programs in LICs. Although we can indeed identify rare, chemotherapy-resistant, primitive LICs, we also observe LICs emerging from more differentiated populations. These facultative LICs self-renew and possess the capability to replenish the full cellular diversity of MLL-r B-ALL, including the primitive populations that retain multipotency programs. In mechanistic studies, we find that the LICs that drive this bottom-up reconstitution of the leukemic cellular ontogeny bear signatures of MYC activation and oxidative phosphorylation. We confirm recruitment of these pathways in actively reconstituting, phenotypically differentiated LICs, and define a pathway by which MYC rewires metabolism in MLL-r B-ALL. We find that MYC is required for LIC plasticity in vitro and in vivo. We conclude that the high LIC content and dual lineage and LIC plasticities of MLL-r B-ALL contribute to its chemotherapy resistance and persistently poor outcomes.

Rowe and colleagues show that hematopoietic cells with MLL translocations undergoing leukemogenesis in neonatal mice yield mixed-lineage early B-lymphoid/myeloid leukemia while identical cells in adults generate myeloid leukemia, demonstrating that the age of the hematopoietic microenvironment impacts lineage., Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

Modeling of Fanconi anemia (FA) using human induced pluripotent stem cells (iPSCs) has been hindered by the requirement for an intact FA DNA repair pathway for effective reprogramming and maintenance of pluripotency in patient-derived iPSCs. Temporary complementation of FA pathway defects could permit effective reprogramming, with removal of complementation upon directed differentiation to hematopoietic stem and progenitor cells (HSPCs) to model the hematopoietic phenotypes of FA. In this study, we used three FA patient-derived iPSC lines engineered with doxycycline inducible complementation of disease-causing FANCA mutations. We maintained complementation with doxycycline exposure in the pluripotent state and during morphogen directed differentiation of hemogenic endothelium (HE) within embryoid bodies, which possesses the capacity to differentiate to HSPCs. Upon initiation of the endothelial-to-hematopoietic transition (EHT) in purified iPSC-derived HE, doxycycline was either removed or maintained in culture to either inactivate or sustain FANCA expression. Morphologic EHT and emergence of CD34+CD45+ immunophenotypic human HSPCs were not affected by the status of FANCA expression, which allowed for the isolation of otherwise isogenic FANCA-expressing and FANCA-deficient HSPCs for disease modeling. FANCA-deficient HSPCs were unable to form FANCD2/γH2AX foci relative to FANCA-expressing HSPCs in response to genotoxic stress, confirming impairment of the function of the FA pathway. We found that uncomplimented, FANCA-deficient HSPCs showed impaired cell cycle progression, increased apoptosis, and markedly decreased colony forming activity in methylcellulose compared to complemented, FANCA-expressing HSPCs. Unexpectedly, we also found that FANCA-deficient HSPCs showed accelerated erythroid differentiation compared to cells with an intact FA pathway. RNA sequencing analysis showed enrichment of signatures of normal HSPCs in complemented HSPCs, while uncomplimented HSPCs showed signatures of more mature hematopoietic cells and cells undergoing erythroid differentiation. Together, these findings demonstrate the utility of reversible complementation in modeling FA with patient-derived iPSCs, and provide an inexhaustible source of otherwise isongenic complimented and uncomplimented human FA HSPCs for use in the investigation of new therapies. Disclosures No relevant conflicts of interest to declare.

This report describes concept inventories, specialized assessment instruments that enable educational researchers to investigate student (mis)understandings of concepts in a particular domain. While students experience a concept inventory as a set of multiple-choice items taken as a test, this belies its purpose, its careful development, and its validation. A concept inventory is not intended to be a comprehensive instrument, but rather a tool that probes student comprehension of a carefully selected subset of concepts that give rise to the most common and pervasive mismodelings. The report explains how concept inventories have been developed and used in other STEM fields, then outlines a project to explore the feasibility of concept inventories in the computing field. We use the domain of discrete mathematics to illustrate a suggested plan of action.