Normal Hematopoiesis Research Articles

Antineoplastic therapy affects the in vitro phenotype and functionality of healthy human bone marrow-derived mesenchymal stromal cells.

While antineoplastic therapies aim to specifically target cancer cells, they may also exert adverse effects on healthy tissues, like healthy hematopoietic stem and progenitor cells (HSPC), leading to hematotoxicity as a common side effect. Mesenchymal stromal cells (MSC) are a major component of the bone marrow (BM) microenvironment, regulating normal hematopoiesis, while their susceptibility to anticancer therapies and contribution to therapy-related hematotoxicity remains largely unexplored. To address this, we investigated the effects of etoposide, temozolomide, 5-azacitidine, and venetoclax on healthy BM-derived MSC functionality. Doses below therapeutic effects of etoposide (0.1-0.25µM) inhibited cellular growth and induced cellular senescence in healthy MSC, accompanied by an increased mRNA expression of CDKN1A, decreased trilineage differentiation capacity, and insufficient hematopoietic support. Pharmacological doses of 5-azacitidine (2.5µM) shifted MSC differentiation capacity by inhibiting osteogenic capacity but enhancing the chondrogenic lineage, as demonstrated by histochemical staining and on mRNA level. At the highest clinically relevant dose, neither venetoclax (40nM) nor temozolomide (100µM) exerted any effects on MSC but clearly inhibited cellular growth of cancer cell lines and primary healthy HSPC, pointing to damage to hematopoietic cells as a major driver of hematotoxicity of these two compounds. Our findings show that besides HSPC, also MSC are sensitive to certain antineoplastic agents, resulting in molecular and functional alterations that may contribute to therapy-related myelosuppression. Understanding these interactions could be helpful for the development of strategies to preserve BM MSC functionality during different kinds of anticancer therapies.

Inhibition of the Notch signal transducer CSL by Pkc53E-mediated phosphorylation to fend off parasitic immune challenge in Drosophila.

Notch signalling activity regulates hematopoiesis in Drosophila and vertebrates alike. Parasitoid wasp infestation of Drosophila larvae, however, requires a timely downregulation of Notch activity to allow the formation of encapsulation-active blood cells. Here, we show that the Drosophila CSL transcription factor Suppressor of Hairless [Su(H)] is phosphorylated at Serine 269 in response to parasitoid wasp infestation. As this phosphorylation interferes with the DNA binding of Su(H), it reversibly precludes its activity. Accordingly, phospho-deficient Su(H)S269A mutants are immune-compromised. A screen for kinases involved in Su(H) phosphorylation identified Pkc53E, required for normal hematopoiesis as well as for parasitoid immune response. Genetic and molecular interactions support the specificity of the Su(H)-Pkc53E relationship. Moreover, phorbol ester treatment inhibits Su(H) activity in vivo and in human cell culture. We conclude that Pkc53E targets Su(H) during parasitic wasp infestation, thereby remodelling the blood cell population required for wasp egg encapsulation.

Chromatin structure and 3D architecture define the differential functions of PU.1 regulatory elements in blood cell lineages

The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

Single-cell transcriptional mapping reveals genetic and non-genetic determinants of aberrant differentiation in AML.

In acute myeloid leukemia (AML), genetic mutations distort hematopoietic differentiation, resulting in the accumulation of leukemic blasts. Yet, it remains unclear how these mutations intersect with cellular origins and whether they converge upon similar differentiation patterns. Single-cell RNA sequencing (scRNA-seq) has enabled high-resolution mapping of the relationship between leukemia and normal cell states, yet this application is hampered by imprecise reference maps of normal hematopoiesis and small sample sizes among patient cohorts. As a first step we constructed a reference atlas of human bone marrow hematopoiesis from 263,519 single-cell transcriptomes spanning 55 cellular states, that was benchmarked against independent datasets of immunophenotypically pure hematopoietic stem and progenitor cells. Using this reference atlas, we mapped over 1.2 million single-cell transcriptomes spanning 318 AML, mixed phenotype acute leukemia (MPAL), and acute erythroid leukemia (AEL) samples. This large-scale analysis, together with systematic mapping of genotype-to-phenotype associations between driver mutations and differentiation landscapes, revealed convergence of diverse genetic alterations on twelve recurrent patterns of aberrant differentiation in AML. This included unconventional lymphoid and erythroid priming linked to RUNX1 and TP53 mutations, respectively. We also identified non-genetic determinants of AML differentiation such as two subgroups of KMT2A-rearranged AML that differ in the identity of their leukemic stem cells (LSCs), likely reflecting distinct cellular origins. Furthermore, distinct LSC-driven hierarchies can co-exist within individual patients, providing insights into AML evolution. Together, precise mapping of normal and malignant cell states provides a framework for advancing the study and disease classification of hematologic malignancies thereby informing therapy development.

FLAER Revealed Normally Expected Non-PNH FLAER-Dim Immature Myeloid Cells (CD117+/CD34-) In Bone Marrow Aspirates and Could Be Utilized as a Marker of Hierarchical Hematopoiesis.

Fluorescently labeled aerolysin (FLAER) is widely used for the identification of paroxysmal nocturnal hemoglobinuria (PNH) clones in peripheral blood (PB) samples. However, there are only a few reports on the differential fluorescent intensity of FLAER in normal bone marrow (BM) cell subpopulations. The purpose of this study was to evaluate FLAER expression during normal and pathological hematopoiesis, to map the critical existence of non-PNH FLAER-dim cells. A total of 54 BM aspirates were prospectively analyzed with FLAER-based flow cytometric (FC) protocols, during their routine work-up. These were obtained from patients with the following diagnoses: PNH (3), infections/reactive (5), myelodysplastic syndromes (MDS) (7), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (4), chronic myelogenous leukemia (CML) (3), acute myelogenous leukemia (AML) at diagnosis (20), AML in measurable residual disease (MRD) assessment (7), and B-cell acute lymphoblastic leukemia (B-ALL) in MRD assessment (5). The applied protocols consisted of FLAER, HLA-DR, CD14, CD33, CD34, CD66b, CD38, CD117, CD64, CD45, and FLAER, CD66c, CD14, CD33, CD34, CD66b, CD123, CD16, CD64, and CD45, respectively. FLAER expression was assessed in CD34++/CD38- and CD34+/CD38+ stem cells, CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, and CD64+/CD14-/HLA-DR+ monocyte precursors but also in mature myeloid cells. All patients revealed an intermediate FLAER intensity in CD34++/CD38- stem cells, with a discrete FLAER-negative subpopulation observed only in maturing CD34+/CD38+ stem cells of patients with PNH. The lowest FLAER intensity was noticed in CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, not only in patients with PNH but also in PNH-negative BM aspirates. An ascending FLAER intensity was further observed during monocytic and granulocytic maturation, with a discrete FLAER-negative population in CD64+/CD14-/HLA-DR+ monocyte precursors and maturing neutrophils and monocytes of patients with PNH only. The maturation pattern of FLAER expression was further confirmed in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), where FLAER was concurrently upregulated with CD66b in a consecutive series of PB samples tested over a 20-day-period after diagnosis. The application of FLAER in PNH-positive and PNH-negative reactive or malignant BM aspirates identified normally expected non-PNH FLAER-dim CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors in all samples. A specific FLAER-associated maturation pattern was observed, which is proposed for further study within MRD and diagnostic protocols.

RNA modification in normal hematopoiesis and hematologic malignancies.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells. Previous studies have shown that m6A plays a critical role under both normal physiological and pathological conditions. Hematopoiesis and differentiation are highly regulated processes, and recent studies on m6A mRNA methylation have revealed how this modification controls cell fate in both normal and malignant hematopoietic states. However, despite these insights, a comprehensive understanding of its complex roles between normal hematopoietic development and malignant hematopoietic diseases remains elusive. This review first provides an overview of the components and biological functions of m6A modification regulators. Additionally, it highlights the origin, differentiation process, biological characteristics, and regulatory mechanisms of hematopoietic stem cells, as well as the features, immune properties, and self-renewal pathways of leukemia stem cells. Last, the article systematically reviews the latest research advancements on the roles and mechanisms of m6A regulatory factors in normal hematopoiesis and related malignant diseases. More importantly, this review explores how targeting m6A regulators and various signaling pathways could effectively intervene in the development of leukemia, providing new insights and potential therapeutic targets. Targeting m6A modification may hold promise for achieving more precise and effective leukemia treatments.

Incidence of myelosuppression in AML is higher compared with that in ALL.

Acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) are two subtypes of acute leukemia. However, studies investigating the ability of complete blood count (CBC) parameters to distinguish between patients with AML and ALL remain scarce in the literature. The objective of the present study was to compare the parameters of CBC analysis between Chinese patients with AML and ALL and between patients with M3 AML and non-M3 AML. Prognostic factors for overall survival were also estimated, including sex, age, white blood cell count and hemoglobin. The present study included 147 patients, including children and adults, with newly diagnosed acute leukemia. Information on the age, sex, leukemia subtype, initial CBC results and clinical follow-up findings was recorded and compared between the indicated groups using statistical tests of Mann-Whitney U test and χ2 test. Leukopenia (white blood cell count <3.5x109/l), both leukopenia and anemia, both leukopenia and thrombocytopenia and pancytopenia were found to be significantly more frequent among patients with AML compared with that in patients with ALL (P=0.015, 0.016, 0.015 and 0.019, respectively). For patients with ALL, anemia was recognized as a predictor of a favorable outcome (Hazard ratio, 0.185; 95% CI, 0.046-0.747; P=0.018). These findings suggest that normal hematopoiesis is more frequently inhibited in patients with AML compared with that in patients with ALL. Patients with AL with peripheral blood findings indicative of leukopenia, pancytopenia, or both leukopenia and anemia or both leukopenia and thrombocytopenia are more likely to have AML.

YTHDC1-mediated microRNA maturation is essential for hematopoietic stem cells maintenance

YTHDC1, a reader of N6-methyladenosine (m6A) modifications on RNA, is posited to exert significant influence over RNA metabolism. Despite its recognized importance, the precise function and underlying mechanisms of YTHDC1 in the preservation of normal hematopoietic stem cell (HSCs) homeostasis remain elusive. Here, we investigated the role of YTHDC1 in normal hematopoiesis and HSCs maintenance in vivo. Utilizing conditional Ythdc1 knockout mice and Ythdc1/Mettl3 double knockout mice, we demonstrated that YTHDC1 is required for HSCs maintenance and self-renewal by regulating microRNA maturation. YTHDC1 deficiency resulted in HSCs apoptosis. Furthermore, we uncovered that YTHDC1 interacts with HP1BP3, a nuclear RNA binding protein involved in microRNA maturation. Deletion of YTHDC1 brought about significant alterations in microRNA levels. However, over-expression of mir-125b, mir-99b, and let-7e partially rescued the functional defect of YTHDC1-null HSCs. Taken together, these findings indicated that the nuclear protein YTHDC1-HP1BP3-microRNA maturation axis is essential for the long-term maintenance of HSCs.

A Diagnostic Challenge in the Biopsy of a Mammographic Abnormality

Abstract Introduction/Objective Primary breast marginal zone lymphoma (MZL) is an uncommon subtype of extranodal lymphoma, and its diagnosis presents challenges, particularly in distinguishing it from lymphoplasmacytic lymphoma (LPL). MYD88 L265P mutation has been identified in a subset of marginal zone lymphomas, comprising 6-9% of cases. This genetic alteration further complicates the differentiation between MZL and LPL in breast tumors. We present a case highlighting the diagnostic complexities associated with primary breast MZL. Methods/Case Report A 71-year-old female patient underwent mammographic screening, which revealed a suspicious lesion in her left breast. Subsequent PET scan showed a hypermetabolic area consistent with a primary breast lesion. Biopsy histology exhibited a diffuse atypical lymphoid infiltration with varying proportions of neoplastic lymphocytes. Immunohistochemical analysis revealed positivity for CD20, PAX5, BCL2, CD138, MUM1, and negativity for CD3, CD5, Cyclin D1, CD10, CD23, CD34, BCL6, TDT, CD30, GATA3, and CK AE1/3. Additionally, there was lambda light chain restriction, and the Ki67 proliferation rate index was approximately 50%. Fish analysis indicated abnormal BCL6 gene rearrangement. Molecular genetic testing confirmed the presence of MYD88 mutation with c.794T&amp;gt;C p.L265P. Notably, bone marrow biopsy showed no abnormalities, with normal hematopoiesis. Serum electrophoresis and immunofixation detected an IgM monoclonal protein with lambda light chain specificity. Results (if a Case Study enter NA) NA Conclusion The diagnosis of primary breast MZL was favored over LPL due to the absence of common LPL- associated symptoms such as bone marrow involvement. However, the distinction between these entities remains challenging, underscoring the importance of comprehensive diagnostic approaches integrating histopathology, immunohistochemistry, and molecular genetics. Further research is warranted to enhance diagnostic certainty in such cases, particularly as the management of MZL and LPL becomes increasingly specific with the advent of novel therapeutic agents and ongoing clinical trials.

Cyclin C promotes development and progression of B-cell acute lymphoblastic leukemia by counteracting p53-mediated stress responses.

Despite major therapeutic advances in the treatment of acute lymphoblastic leukemia (ALL), resistances and long-term toxicities still pose significant challenges. Cyclins and their associated cyclin-dependent kinases are one focus of cancer research when looking for targeted therapies. We discovered cyclin C as a key factor for B-ALL development and maintenance. While cyclin C is non-essential for normal hematopoiesis, CcncΔ/Δ BCR::ABL1+ B-ALL cells fail to elicit leukemia in mice. RNA sequencing experiments revealed a p53 pathway deregulation in CcncΔ/Δ BCR::ABL1+ cells resulting in the incapability of the leukemic cells to adequately respond to stress. A genome-wide CRISPR/Cas9 loss-of-function screen supplemented with additional knock-outs unveiled a dependency of human B-lymphoid cell lines on CCNC. High cyclin C levels in B-cell precursor (BCP) ALL patients were associated with poor event-free survival and increased risk of early disease recurrence after remission. Our findings highlight cyclin C as potential therapeutic target for B-ALL, particularly to enhance cancer cell sensitivity to stress and chemotherapy.

The small inhibitor WM-1119 effectively targets KAT6A-rearranged AML, but not KMT2A-rearranged AML, despite shared KAT6 genetic dependency

BackgroundThe epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors’ self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs.MethodsWe investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs.ResultsWe found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML.ConclusionOur study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs.

7686 A Case Of A 66-Year-Old Male With Systemic Macrocytosis Complicated By Osteoporosis

Abstract Disclosure: J.M. Gri: None. M. Hillhouse: None. P. madhavan: None. Background: Osteoporosis in men is underestimated and warrants investigation for secondary causes of osteoporosis. Systemic mastocytosis is a rare secondary cause of osteoporosis. Clinical Case: This is a 66-year-old male with a past medical history of severe persistent asthma, eczema, hypertension, GERD with esophagitis, basal cell carcinoma, and systemic mastocytosis (diagnosed in 2010). Family history was significant for lung cancer in both of his parents. Patient was first noted to have hyperpigmented skin lesions on his legs, trunk and upper extremities in the early 1990s. The spots were biopsied and he was diagnosed with urticaria pigmentosa in 1998. Due to persistent symptoms of facial flushing and soft stool, he was referred to Hematology and was started on antihistamines. He later underwent a bone marrow biopsy in 2010 which showed multifocal mast cell aggregates but normal trilineage hematopoiesis. He endorsed back pain shortly after his diagnosis and a DXA scan was completed. The DXA scan showed osteopenia of the spine T score of -1.8, BMD 0.999 mg/mm3 and normal bone density in bilateral hips. He had no history of fractures. He was started on alendronate and remained on the medication from 2012 to 2015. It was stopped due to intolerable symptoms of acid reflux and he required proton pump inhibitors. DXA scan in 2017 showed normal bone density of the hips and osteopenia of the lumbar spine (T score -1.2). DXA scan in 2022 showed a decrease in spine BMD by 6% (T score -1.8) from 2017 to 2022 but unchanged from 2010. Hip bone mineral density was normal but decreased by 10% since 2010 and by 6% since 2017. Labs obtained in May 2022 showed normal serum electrophoresis, normal urine calcium and creatinine ratio, calcium and vitamin D level and urine NTX of 41 nM BCE/mM (21-83) and bone specific alkaline phosphatase level of 31.5 U/L (15-41.3). After discussion with the patient, conservative management with calcium, vitamin D and exercise has been implemented along with close follow up. Should the patient’s bone turnover markers increase or fracture risk increase, additional antiresorptive therapy will be initiated. Conclusion: This case study highlights the importance of screening for osteoporosis in patients with systemic macrocytosis. As this patient was diagnosed with osteopenia at age 53, this case also emphasizes the importance of early screening in both women and men with this rare condition. Presentation: 6/3/2024

Relationship between adipocytes and hematological tumors in the bone marrow microenvironment: a literature review.

The bone marrow microenvironment is closely related to normal hematopoiesis and hematologic tumors. Adipocytes are an important part of the bone marrow microenvironment, in which they can release free fatty acids (FFAs) through lipolysis and secrete adipocytokines, etc., and participate in normal hematopoiesis, which is closely related to the occurrence and treatment of hematological tumors. In this review, we aim to discuss how bone marrow adipocytes (BMAs) can influence the proliferation, apoptosis, and chemotherapy resistance of cancer cells by reprogramming lipid metabolism and the secretion of various adipocytokines. Studies from 2000 to July 2024 were reviewed from PubMed, Springer Link, and the Web of Science using the keywords bone marrow microenvironment, adipocytes, lipid metabolism, adipocytokines, hematological tumor, cancer, and their combinations. Unreliable articles such as those that are old and have a low impact factor are excluded, and there is no restriction on language. Adipocytes can regulate the proliferation and differentiation of hematopoietic stem cells (HSCs) by secreting inflammatory factors and adipocytokines to maintain hematopoietic homeostasis. Adipocytes can also stimulate and accelerate the occurrence and progression of hematological tumors by secreting adipocytokines and mediating the reprogramming of lipid metabolism. Moreover, abundant adipocytes in bone marrow can protect tumor cells by physically blocking and/or secreting cytokines, leading to chemotherapy resistance. Therefore, the targeted inhibition of related lipid metabolism pathways and adipocytokines might be a potential therapeutic target for hematological tumors, which would be helpful to inhibit tumor growth and correct chemotherapy resistance.

Pharmacological targeting of CBX7 alters the epigenetic landscape and induces differentiation of leukemic cells

AbstractSelf-renewal of leukemic cells results in the accumulation of dysfunctional blood cells and suppression of normal hematopoiesis. The polycomb group protein chromobox 7 (CBX7) is an epigenetic regulator that represses genes required for differentiation and cell cycle arrest and thereby promotes self-renewal. Because leukemic cells are highly self-renewing, we tested whether pharmacological targeting of CBX7 would reduce self-renewal and induce differentiation of human leukemic cells. We found that existing and newly developed CBX7 inhibitors derepress the epigenome, resulting in reduced ubiquitination of histone 2A and reduced binding of CBX7 to its target genes. This led to reduced cell growth, increased differentiation of leukemic cells in vitro, and delayed engraftment of primary leukemic cells in xenotransplant models. Therefore, pharmacological targeting of CBX7 constitutes a novel therapeutic approach for leukemia.

Inhibition of TGF-β signaling in bone marrow endothelial cells promotes hematopoietic recovery in acute myeloid leukemia patients

Although it is an effective treatment for acute myeloid leukemia (AML), chemotherapy leads to myelosuppression and poor hematopoietic reconstruction. Hematopoiesis is regulated by bone marrow (BM) endothelial cells (ECs), and BM ECs are dysfunctional in acute leukemia patients with poor hematopoietic reconstitution after allogenic hematopoietic stem cell transplantation. Thus, it is crucial to explore the underlying mechanism of EC impairment and establish strategies for targeted therapy. TGF-β signaling was found to be upregulated in ECs from AML patients in complete remission (CR ECs) and led to CR EC damage. Administration of a TGF-β inhibitor rescued the dysfunction of ECs caused by TGF-β1 expression in vitro, especially their hematopoiesis-supporting ability. Moreover, inhibition of TGF-β expression repaired the BM EC damage triggered by chemotherapy in both AML patients in vitro and in an AML-CR murine model, and restored normal hematopoiesis without promoting AML progression. Mechanistically, our data reveal alterations in the transcriptomic pattern of damaged BM ECs, accompanied by the overexpression of downstream molecules TGF-βRI, pSmad2/3, and functional genes related to adhesion, angiogenesis suppression and pro-apoptosis. Collectively, our findings reveal for the first time that the activation of TGF-β signaling leads to BM EC dysfunction and poor hematopoietic reconstitution. Targeting TGF-β represents a potential therapeutic strategy to promote multilineage hematopoiesis, thereby benefiting more cancer patients who suffer from myelosuppression after chemotherapy.

Mycovirus-Containing Aspergillus flavus Alters Transcription Factors in Normal and Acute Lymphoblastic Leukemia Cells.

Transcription factors control genes to maintain normal hemopoiesis, and dysregulation of some factors can lead to acute lymphoblastic leukemia (ALL). Mycoviruses are known to alter the genetics of their fungal host. The present study evaluates the effects of the products of a mycovirus-containing Aspergillus flavus (MCAF), isolated from the home of a patient with ALL, on certain transcription factors of normal and ALL cell lines. Our published studies have shown that ALL patients have antibodies to MCAF, and that exposure of the mononuclear leukocytes of patients in complete remission to its products, unlike controls, results in the re-development of genetic and cell surface phenotypes characteristic of ALL. For the present study, normal, pre-B, and B-cell leukemia cell lines were exposed to the culture of MCAF. Pre- and post-exposure levels of PAX5, Ikaros, and NF-κB were assessed. Exposure to MCAF resulted in apoptosis, cell cycle changes, and complete downregulation of all transcription factors in normal cell lines. In acute leukemia cell lines, cellular apoptosis and alterations in the cell cycle were also noted; however, while there was downregulation of all tested transcription factors, residual levels were retained. The noted alterations in the transcription factors caused by MCAF are novel findings. The possible role of MCAF in leukemogenesis needs to be further investigated. Mycovirus-containing Aspergillus flavus was initially isolated from a leukemia patient's home. Our prior published studies have illuminated intriguing associations of this organism with leukemia. Unlike controls, patients diagnosed with acute lymphoblastic leukemia (ALL) harbor antibodies to this organism. Furthermore, the exposure of mononuclear cells from patients with ALL in complete remission to the products of this organism reproduced genetic and cell phenotypes characteristic of ALL. These findings underscore the potential role of environmental factors in leukemogenesis and hint at novel avenues for therapeutic intervention and preventive strategies.

Hydroxyurea: An Old Drug in Need of New Clinical Trials in Myeloproliferative Neoplasms?

Hydroxyurea (Hu) has been a front-line therapeutic agent for myeloproliferative neoplasms (MPN) for many years and still enjoys the endorsement of experts in the field. However, several publications have reported sub-optimal response, the need for treatment interruption because of cytopenias and lack of sustained response. In all these studies, Hu was used as continuous therapy at a daily dose ranging from 500mg to 3000mg. At our Centre we have used Hu as intermittent therapy (akin to schedules used in patients with solid tumours) at 20-30mg/Kg doses, given as a single dose, twice or thrice weekly. We have consistently observed sustained responses without troublesome cytopenias. Concurrent anti-platelet therapy was given on the basis of the results of whole blood platelet aggregation studies, achieving effective thromboprophylaxis. In this report we present our experience in 118 patients treated with intermittent Hu during the past 30 years (median follow-up 8.5 yrs): polycythemia vera – 29; essential thrombocythemia – 84; primary myelofibrosis – 5. Based on the pharmacokinetics of Hu and our experience, we speculate that the efficacy of intermittent Hu therapy without troublesome myelotoxicity over long periods of time is attributable to the following: i) higher plasma level from intake of Hu as a single dose; ii) higher uptake of Hu by cells with higher mitotic activity (i.e. the abnormal clone); and iii) unhindered, normal haemopoiesis on the drug free days each week. We hope that this article will generate interest and contemplation, leading to further publications from Centres using intermittent Hu therapy and randomized clinical studies to compare the two dosage schedules (Continuous Vs Intermittent) in MPN patients.

Selective targeting of mutated calreticulin by the monoclonal antibody INCA033989 inhibits oncogenic function of MPN

AbstractMutations in calreticulin (mutCALR) are the second most common drivers of myeloproliferative neoplasms (MPNs) and yet, the current therapeutic landscape lacks a selective agent for mutCALR-expressing MPNs. Here, we show that the monoclonal antibody INCA033989 selectively targets mutCALR-positive cells. INCA033989 antagonized mutCALR-driven signaling and proliferation in engineered cell lines and primary CD34+ cells from patients with MPN. No antibody binding or functional activity was observed in the cells lacking mutCALR. In a mouse model of mutCALR-driven MPN, treatment with an INCA033989 mouse surrogate antibody effectively prevented the development of thrombocytosis and accumulation of megakaryocytes in the bone marrow. INCA033989 reduced the pathogenic self-renewal of mutCALR-positive disease-initiating cells in both primary and secondary transplantations, illustrating its disease-modifying potential. In summary, we describe a novel mutCALR-targeted therapy for MPNs, a monoclonal antibody that selectively inhibits the oncogenic function of MPN cells without interfering with normal hematopoiesis.

Cohesin mutations in acute myeloid leukemia.

The cohesin complex, encoded by SMC3, SMC1A, RAD21, and STAG2, is a critical regulator of DNA-looping and gene expression. Over a decade has passed since recurrent mutations affecting cohesin subunits were first identified in myeloid malignancies such as Acute Myeloid Leukemia (AML). Since that time there has been tremendous progress in our understanding of chromatin structure and cohesin biology, but critical questions remain because of the multiple critical functions the cohesin complex is responsible for. Recent findings have been particularly noteworthy with the identification of crosstalk between DNA-looping and chromatin domains, a deeper understanding of how cohesin establishes sister chromatid cohesion, a renewed interest in cohesin's role for DNA damage response, and work demonstrating cohesin's importance for Polycomb repression. Despite these exciting findings, the role of cohesin in normal hematopoiesis, and the precise mechanisms by which cohesin mutations promote cancer, remain poorly understood. This review discusses what is known about the role of cohesin in normal hematopoiesis, and how recent findings could shed light on the mechanisms through which cohesin mutations promote leukemic transformation. Important unanswered questions in the field, such as whether cohesin plays a role in HSC heterogeneity, and the mechanisms by which it regulates gene expression at a molecular level, will also be discussed. Particular attention will be given to the potential therapeutic vulnerabilities of leukemic cells with cohesin subunit mutations.

The small noncoding RNA Vaultrc5 is dispensable to mouse development.

Vault RNAs (vtRNAs) are evolutionarily conserved small noncoding RNAs transcribed by RNA polymerase III. Vault RNAs were initially described as components of the vault particle, but have since been assigned multiple vault-independent functions, including regulation of PKR activity, apoptosis, autophagy, lysosome biogenesis, and viral particle trafficking. The full-length transcript has also been described as a noncanonical source of miRNAs, which are processed in a DICER-dependent manner. As central molecules in vault-dependent and independent processes, vtRNAs have been attributed numerous biological roles, including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss-of-function allele. Because Vaultrc5 is the sole murine vtRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts, pointing to a potential role for vtRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.