Haematologica

Fanconi anemia is a rare inherited bone marrow failure syndrome caused by inactivation of genes in the Fanconi anemia/BRCA DNA repair pathway. We report a patient with X-linked Fanconi anemia, and atypical physical features whose genetic diagnosis was initially inconclusive. Over time, his bone marrow karyotype shifted from diploid (46,XY) to triploid (69,XXY). The triploid cells lacked the Fanconi anemia cellular phenotype, enabling sustained hematopoiesis and providing an unexpected route to phenotypic rescue. Genomic analysis indicated early post-zygotic incorporation of the second polar body as the triploid origin. These findings suggest that the selective advantage of restored DNA repair in hematopoietic stem cells, outweigh the potentially deleterious effects of triploidy.

Measurable residual disease (MRD) in adults with acute myeloid leukemia (AML) in complete remission is an important prognostic marker, but detection methodology requires optimization. The persistence of mutated NPM1 or FLT3-ITD in the blood of adult patients with AML in first complete remission (CR1) prior to allogeneic hematopoetic cell transplant (alloHCT) has been established as associated with increased relapse and death after transplant. The prognostic implications of persistence of other common AML-associated mutations, such as IDH1, at this treatment landmark however remains incompletely defined. We performed testing for residual IDH1 variants (IDH1m) in pre-transplant CR1 blood of 148 adult patients undergoing alloHCT for IDH1-mutated AML at a CIBMTR site between 2013-2019. No post-transplant differences were observed between those testing IDH1m positive (n=53, 36%) and negative pre-transplant (overall survival: p = 0.4; relapse: p = 0.5). For patients with IDH1 mutated AML co-mutated with NPM1 and/or FLT3-ITD, only detection of persistent mutated NPM1 and/or FLT3-ITD was associated with significantly higher rates of relapse (p = 0.01). These data, from the largest study to date, do not support the detection of IDH1 mutation in CR1 blood prior to alloHCT as evidence of AML MRD or increased post-transplant relapse risk.

The GATA2 transcription factor is a pivotal regulator of hematopoiesis. Disruptions in the GATA2 gene drive severe hematologic abnormalities and are associated with an increased risk of myelodysplastic syndromes and acute myeloid leukemia; however, the mechanisms underlying the pathophysiology of GATA2 deficiency remain still unclear. We developed two different mouse models that are based on serial and limiting donor cell transplantation of (aged) GATA2 haploinsufficient cells and mirror the symptoms of GATA2 deficiency. Similar to what has been observed in patients, our models show that GATA2 haploinsufficiency leads to B lymphopenia, monocytopenia, lethal bone marrow failure (BMF), myelodysplasia and leukemia. Leukemia arises exclusively as a result of BMF, driven by somatic aberrations and accompanied by increased Myc target expression and genomic instability. These findings were confirmed in human GATA2+/- K562 cell lines showing defects in cytokinesis and are in line with the fact that monosomy 7 and trisomy 8 are frequent events in patients with MDS. Key pointsO_LIIn a mouse model for GATA2 deficiency, leukemia emerges from bone marrow failure C_LIO_LIMaladaptation to proliferative signals and chromosomal segregation defects contribute to the hematological phenotypes in GATA2 deficiency C_LI

Mutations commonly found in AML such as DNMT3A, TET2 and ASXL1 can be found in the peripheral blood of otherwise healthy adults - a phenomenon referred to as clonal hematopoiesis (CH). These mutations are thought to represent the earliest genetic events in the evolution of AML. Genomic studies on samples acquired at diagnosis, remission, and at relapse have demonstrated significant stability of CH mutations following induction chemotherapy. Meanwhile, later mutations in genes such as NPM1 and FLT3, have been shown to contract at remission and in the case of FLT3 often are absent at relapse. We sought to understand how early CH mutations influence subsequent evolutionary trajectories throughout remission and relapse in response to induction chemotherapy. Here, we assembled a retrospective cohort of patients diagnosed with de novo AML at our institution that underwent genomic sequencing at diagnosis as well as at the time of remission and/or relapse (total n = 182 patients). Corroborating prior studies, FLT3 and NPM1 mutations were generally eliminated at the time of cytologic complete remission but subsequently reemerged upon relapse, whereas DNMT3A, TET2 and ASXL1 mutations often persisted through remission. Early CH-related mutations exhibited distinct constellations of co-occurring genetic alterations, with NPM1 and FLT3 mutations enriched in DNMT3Amut AML, while CBL and SRSF2 mutations were enriched in TET2mut and ASXL1mut AML, respectively. In the case of NPM1 and FLT3 mutations, these differences vanished at the time of complete remission yet readily reemerged upon relapse, indicating the reproducible nature of these genetic interactions. Thus, early CH-associated mutations that precede malignant transformation subsequently shape the evolutionary trajectories of AML through diagnosis, therapy, and relapse. Key PointsO_LIDNMT3A, TET2 and ASXL1 mutations persist through AML-directed therapy C_LIO_LIDistinct CH-related mutations shape the evolutionary trajectories of AML from diagnosis through relapse. C_LI

Polygenic variation unrelated to disease contributes to interindividual variation in baseline white blood cell (WBC) counts, but its clinical significance is undefined. We investigated the clinical consequences of a genetic predisposition toward lower WBC counts among 89,559 biobank participants from tertiary care centers using a polygenic score for WBC count (PGSWBC) comprising single nucleotide polymorphisms not associated with disease. A predisposition to lower WBC counts was associated with a decreased risk of identifying pathology on a bone marrow biopsy performed for a low WBC count (odds-ratio=0.55 per standard deviation increase in PGSWBC [95%CI, 0.30 - 0.94], p=0.04), an increased risk of leukopenia (a low WBC count) when treated with a chemotherapeutic (n=1,724, hazard ratio [HR]=0.78 [0.69 - 0.88], p=4.0x10-5) or immunosuppressant (n=354, HR=0.61 [0.38 - 0.99], p=0.04). A predisposition to benign lower WBC counts was associated with an increased risk of discontinuing azathioprine treatment (n=1,466, HR=0.62 [0.44 - 0.87], p=0.006). Collectively, these findings suggest that a WBC count polygenic score identifies individuals who are susceptible to escalations or alterations in clinical care that may be harmful or of little benefit.

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system.1 Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs.2 The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro. Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41+ MK-committed HSCs in vitro. Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenance in vitro. DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.

Telomere biology disorders (TBDs), exemplified by dyskeratosis congenita (DC), are characterized by genetic defects in telomere maintenance genes, leading to telomere attrition and multi-organ manifestations including bone marrow failure (BMF) and increased malignancy risk. This study aimed to evaluate the prevalence and clinical impact of rare possibly significant variations (PSVs) in telomere-related genes among patients with BMF and clonal hematopoiesis disorders. We analyzed 1320 patients diagnosed with aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, or acute lymphoblastic leukemia and identified 113 PSVs in 103 patients, significantly exceeding the prevalence in the general population (gnomAD). The most frequently mutated genes were RTEL1 (37%) and CTC1 (30%). While missense variants predominated, novel variants accounted for approximately 27.4%. Patients harboring PSVs exhibited significantly shorter telomeres compared to unaffected relatives, reinforcing telomere length as a critical functional biomarker. Telomere-mediated genetic anticipation was clearly evident: younger patients had notably shorter telomeres compared to their older first-degree relatives, reflecting cumulative generational telomere attrition and progressively severe phenotypes. Despite variability in clinical presentations--with many patients lacking classical mucocutaneous or fibrotic manifestations--telomere shortening provided robust onset information. Our findings emphasize the importance of integrating genetic testing and telomere length measurement into clinical practice for early diagnosis, personalized risk stratification, and tailored management, including reduced-intensity conditioning transplantation and emerging targeted therapies.

The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within the erythrocyte membrane and is required for glucose and dehydroascorbic acid (Vitamin C precursor) transport. It is widely recognized as a key protein for red cell structure, function, and metabolism. Previous reports highlighted the importance of GLUT1 activity within these uniquely glycolysis-dependent cells, in particular for increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress in humans. However, studies of glucose transporter roles in erythroid cells are complicated by species-specific differences between humans and mice. Here, using CRISPR-mediated gene editing of immortalized erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate committed human erythroid cells completely deficient in expression of GLUT1. We show that absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This work demonstrates for the first-time generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient reticulocytes possess no tangible alterations to membrane composition or deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient reticulocytes reveal hallmarks of reduced glucose import, downregulated metabolic processes and upregulated AMPK-signalling, alongside alterations in antioxidant metabolism, resulting in increased osmotic fragility and metabolic shifts indicative of higher oxidant stress. Despite detectable metabolic changes in GLUT1 deficient reticulocytes, the absence of developmental phenotype, detectable proteomic compensation or impaired deformability comprehensively alters our understanding of the role of GLUT1 in red blood cell structure, function and metabolism. It also provides cell biological evidence supporting clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 deficiency syndrome. Key PointsO_LIGLUT1 knockout does not affect erythroid differentiation and minimally impacts reticulocyte membrane composition C_LIO_LIMetabolic adaptation facilitates reticulocyte tolerance of GLUT1 absence C_LI

TP53-mutated myeloid malignancies are most frequently associated with complex cytogenetics. The presence of complex and extensive structural variants complicates detailed genomic analysis by conventional clinical techniques. We performed whole genome sequencing of 42 AML/MDS cases with paired normal tissue to characterize the genomic landscape of TP53-mutated myeloid malignancies. The vast majority of cases had multi-hit involvement at the TP53 genetic locus (94%), as well as aneuploidy and chromothripsis. Chromosomal patterns of aneuploidy differed significantly from TP53-mutated cancers arising in other tissues. Recurrent structural variants affected regions that include ETV6 on chr12p, RUNX1 on chr21, and NF1 on chr17q. Most notably for ETV6, transcript expression was low in cases of TP53-mutated myeloid malignancies both with and without structural rearrangements involving chromosome 12p. Telomeric content is increased in TP53-mutated AML/MDS compared other AML subtypes, and telomeric content was detected adjacent to interstitial regions of chromosomes. The genomic landscape of TP53-mutated myeloid malignancies reveals recurrent structural variants affecting key hematopoietic transcription factors and telomeric repeats that are generally not detected by panel sequencing or conventional cytogenetic analyses. Key PointsO_LIWGS comprehensively determines TP53 mutation status, resulting in the reclassification of 12% of cases from mono-allelic to multi-hit C_LIO_LIChromothripsis is more frequent than previously appreciated, with a preference for specific chromosomes C_LIO_LIETV6 is deleted in 45% of cases, with evidence for epigenetic suppression in non-deleted cases C_LIO_LINF1 is mutated in 48% of cases, with multi-hit mutations in 17% of these cases C_LIO_LITP53-mutated AML/MDS is associated with altered telomere content compared with other AMLs C_LI

The presence of measurable residual disease (MRD) prior to an allogeneic hematopoietic transplant (alloHCT) in Acute Myeloid Leukemia (AML) has been shown to be associated with an increased risk of post-transplant relapse. Since the Isocitrate Dehydrogenase genes (IDH1/2) are mutated in a considerable proportion of patients with AML, we studied if these mutations would serve as useful targets for MRD. Fifty-five IDH-mutated AML patients undergoing non-myeloablative alloHCT with post-transplant cyclophosphamide at a single center were sequenced at baseline using a multi-gene panel followed by targeted testing for persistent IDH mutations at the pre- and post-alloHCT timepoints by digital droplet PCR or error-corrected next generation sequencing. The cohort included patients who had been treated with IDH inhibitors pre- and post-transplant (20% and 17% for IDH1 and 38% and 28% for IDH2). Overall, 55% of patients analyzed had detectable IDH mutations during complete remission prior to alloHCT. However, there were no statistically significant differences in overall survival (OS), relapse-free survival (RFS), and cumulative incidence of relapse (CIR) at 3 years between patients who tested positive or negative for a persistent IDH mutation during remission (OS: IDH1 p=1, IDH2 p=0.87; RFS: IDH1 p=0.71, IDH2 p= 0.78; CIR: IDH1 p=0.92, IDH2 p=0.97). There was also no difference in the prevalence of persistent IDH mutation between patients who did and did not receive an IDH inhibitor (p=0.59). Mutational profiling of available relapse samples showed that 8 out of 9 patients still exhibited the original IDH mutation, indicating that the IDH mutations remained stable through the course of the disease. This study demonstrates that persistent IDH mutations during remission is not associated with inferior clinical outcomes after alloHCT in patients with AML.

The nuclear oncoprotein SET (patient "SE" translocation) has been implicated in the etiology of MLL/KMT2A-fusion induced leukemia. Here we examine the details of this dependency in murine, primary hematopoietic cells. Experiments demonstrated Set as downstream target of HoxA9 and a direct interactor of Mll/Kmt2A. Mll/Kmt2A and Set globally co-bound promoter regions. Impairing Set expression induced a metabolic shift towards oxidative phosphorylation phenocopying a knockdown of Mll/Kmt2A fusion targets. Set acted predominantly as transcriptional activator driving a pro-proliferative gene expression program with features indicative for Mll/Kmt2A involvement. Molecularly, Set depletion caused dissociation of Mll/Kmt2A from chromatin accompanied by a selective loss of elongating RNA PolymeraseII Ser2-P. Concomitant with a function of Set as inhibitor of protein phosphatase 2A (PP2A), specific recruitment of PP2A to the Meis1 promoter, a known Mll/Kmt2A target, inhibited transcription in reporter assays and in a natural chromatin environment. We identified Mitogen and stress induced kinase 1 (Msk1) as potential substrate protected by Set from dephosphorylation. Active and phosphorylated Msk1-P colocalized with Mll and disappeared from chromatin upon Set depletion. Biochemically, Msk-1 bound directly to Mll/Kmt2A as well as to menin, a known Mll/Kmt2a tethering factor. Loss of Set/Mll/Msk1 selectively affected H3K14 acetylation at promoters and this could be partially attributed to the reduced presence of the histone acetyltransferase Moz/Kat6a. Finally, we show that kinase and menin inhibitors cooperate in leukemia cells indicating that the relay function of Mll/Kmt2A, allowing control of hematopoiesis by cellular signaling, is retained in MLL-fusion proteins.

Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect. This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.

BackgroundThe clinical significance of most germline variants in hematological malignancies (HMs) remains unknown. This presents a challenge in the clinical setting, as the inability to accurately detect pathogenic variants can influence therapeutic decisions. Population isolates have been shown to be beneficial in pathogenic variant discovery due to presence of rare deleterious variants in relatively high frequencies. MethodsWe developed and applied PaVaDi, a computational pipeline that follows American College of Medical Genetics and Genomics (ACMG) guidelines, to evaluate the pathogenicity of germline variants in 511 HM patients from the Finnish founder population. We conducted an exome-wide burden analysis to assess the overall contribution of pathogenic variants to HMs and identified significant gene associations. We also examined genes previously associated with hematological diseases and DNA repair in more detail, and performed protein stability analyses to resolve variants of unknown significance (VUS). ResultsThe exome-wide burden analysis revealed potential pathogenic alleles in CUX2, RNPC3, and MFSD2A that have not previously been linked to HM predisposition. We also identified the largest series of CHEK2 variant carriers reported in hematological diseases, including pathogenic/likely pathogenic (P/LP) variants (n=19), Ile200Thr (i.e., Ile157Thr) (n=49), and other variants of uncertain significance (n=3). CHEK2 variants were 1.7-fold enriched in patients compared to controls (13.9% vs 8.3%, p=2x10-5). Strikingly, Ile200Thr was enriched over four-fold in acute lymphoblastic leukemia patients. Finally, protein structure stability analyses suggested novel MPO variants to be potentially highly deleterious. ConclusionsThis study highlights the importance of germline testing in hematological malignancies and demonstrates the utility of population isolates for pathogenic variant discovery. Our findings identify a significant burden of deleterious variants in HM patients, particularly in CHEK2, and underscore the potential of multi-disease joint analyses in revealing germline contributions to hematological diseases.

B cell antigen receptor (BCR) signaling competence is critical for pathogenesis of chronic lymphocytic leukemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKC function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKC{beta}II upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKC-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKC-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKC-KR expressing cells, coincident with upregulation of PKC{beta}II: supported by activation of key signaling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKC{beta}II inhibitor) treatment of PKC-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKC{beta}II in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKC-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKC{beta} expression facilitates leukemogenesis and identify that BCR-mediated signaling is a key driver of CLL development in the PKC-KR model. Statement of SignificancePKC{beta} facilitates leukemogenesis of CLL, driven through an SP1-regulated transcriptional program and promotes BCR signaling. Thus far, PKC{beta} is the only kinase within the BCR signaling pathway, a key pathway in driving CLL pathogenesis, implicated in the generation of neoplastic B lineage cells.

TP53 is the most frequently mutated gene in human cancers. In Acute Myeloid Leukemia (AML) and Clonal Hematopoiesis of Indeterminate Potential (CHIP), it is one of several recurrent genetic alterations. Despite multiple recent therapeutic advances for AML, TP53 mutated AML is associated with resistance to currently approved therapies and thus, a very poor prognosis. Emerging evidence suggests that mutations in TP53 may be a predictor of positive response to immunotherapy. To model cell - extrinsic consequences of hematopoietic p53 loss, we generated bone marrow chimeric mice bearing p53-/- and congenic wild type cells. Following reconstitution, we observed increased levels of wild type CD8+ and CD4+ T cells in mice transplanted with p53-/- hematopoietic cells compared to controls. In addition, we observed a change in the frequency of T cell subsets in p53-/- chimeras including an increase in Tregs. To determine if these alterations were mirrored in the leukemic setting, we next generated p53-/-;nRasG12D leukemia. While the bone marrow of p53-/-;nRasG12D leukemia showed the presence of both T and B lymphocytes, MLL-AF9 showed a near complete absence of lymphocytes, akin to immune-infiltrated and immune-desert phenotypes seen in solid tumors. These data clearly demonstrate a causal cell-extrinsic effect of hematopoietic p53 loss on the immune system, both in the context of leukemia and preleukemic states. Modeling AML genetics in murine models serves as a powerful tool to define the association between genetic drivers and immune subtypes of AML towards precise patient stratification critical for the application of emerging targeted and immune therapies. Statement of SignificanceTP53 mutations are frequent in both AML and CHIP, and are associated with both resistance to therapy as well as very poor prognosis. We provide evidence to investigate the immunotherapy as a treatment option for this subgroup of AML.

Clonal hematopoiesis of indeterminate potential (CHIP) is an asymptomatic condition associated with elevated risk for myeloid neoplasms (MN). Patients with CHIP and cytopenia are at greater risk of MN. Quantifying the incidence of cytopenia and identifying risk factors among CHIP patients is critical for improving clinical management. We analyzed sequencing data from 805,249 participants in the NIH All of Us Research Program (AoU), Vanderbilts BioVU repository, and UK Biobank (UKB). Genetic mutations, laboratory values, and MN diagnoses were included in survival analyses to determine predictors of cytopenia in individuals with CHIP and matched controls. The cohort contained 9,374 CHIP cases and 24,749 controls. Cytopenia occurred in 13.5% of cases and 11.6% of controls (HR = 1.17, 95% confidence interval: 1.10 - 1.25, P=2.5 x 10-6). Cytopenia risk factors included smoking, male sex, variant allele frequency [&ge;] 0.20, age [&ge;] 65, mean corpuscular volume [&ge;] 100 femtoliters, red cell distribution width [&ge;] 15%, mutations in high-risk CHIP genes, and [&ge;] 2 CHIP mutations. In BioVU, 45% of participants with [&ge;] 4 risk factors progressed to cytopenia within two years. Individuals with CHIP and cytopenia progressed to MN at a rate of ~2% per year, compared to <0.1% per year for those without cytopenia. Longitudinal analysis across three cohorts demonstrated an increased risk of cytopenia in CHIP patients and identified those at highest risk. These findings suggest that cytopenia is a critical step in progression from CHIP to MN, underscoring its utility as an endpoint in cancer prevention trials for CHIP patients.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols unable to deplete LSCs, which reconstitute the disease in vivo, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein dynamics and the role of protein degradation in drug resistance and relapse. Here, using a highly specific inhibitor of the SCFSKP2-CKS1 complex, we report a dual role for CKS1-dependent protein degradation in reducing AML blasts in vivo, and importantly depleting LSCs. Whilst many AML LSC targeted therapies show significant toxicity to healthy hematopoiesis, inhibition of CKS1-dependent protein degradation has the opposite effect, protecting normal hematopoietic cells from chemotherapeutic toxicity. Together these findings demonstrate CKS1-dependent proteostasis is key for normal and malignant hematopoiesis. SignificanceCKS1-dependent protein degradation is a specific vulnerability in AML LSCs. Specific inhibition of SCFSKP2-CKS1 is lethal to CKS1Bhigh AML blasts and all AML LSCs. Normal hematopoiesis is protected from chemotherapeutic toxicity by inhibition of CKS1-dependent protein degradation, substantiating a dual role for CKS1-dependent protein degradation in clinical treatment of AML.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a recently discovered autoinflammatory disorder linked to somatic mutations in the UBA1 gene, resulting in a profound cytoplasm-restricted defect in ubiquitylation. The disease is characterized by a macrocytic anemia that remains poorly understood. To investigate the erythroid lineage in VEXAS, we conducted a comprehensive study combining in vivo assessments of patients mature red cells and marrow erythroblasts, alongside in vitro base-editing models of erythropoiesis. Here we show that mature red cells do not exhibit ubiquitylation defects, and patient-derived bone marrow erythroblasts lack UBA1 mutations beyond the basophilic stage of erythroid differentiation. In vitro base editing of UBA1 variants in CD34+ primary cells resulted in high mortality during early erythroid differentiation, but not during monocytic differentiation. Edited erythroid precursors displayed TP53 overexpression linked to defective ubiquitylation and anomalies in ribosome biogenesis, reminiscent of Diamond-Blackfan anemia. We propose that VEXAS-associated anemia should be considered as a mosaic erythroblastopenia, where the severity of anemia is influenced by the quality and quantity of the UBA1-WT compartment. These insights may aid clinicians in tailoring treatment strategies.

Non-canonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow and tonsil donors, performed RNA-seq, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a cancer driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript utilized an alternative first exon to produce the poorly characterized FBXW7{beta} isoform, previously thought to be restricted to neural tissues. The FBXW7{beta} transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the bone marrow. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7{beta} mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7{beta}, we utilized gain- and loss-of-function approaches and identified an FBXW7{beta}-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7{beta} expression. This signature contained several members of the TNF superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7{beta} expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis and responses to anti-cancer therapies. Key pointsO_LIPreviously thought to be restricted to neural tissues, FBXW7{beta} is the predominant FBXW7 isoform in normal and malignant human pro-B cells. C_LIO_LIFBXW7{beta} promotes transcriptional activation of a proinflammatory gene cluster that contains TNF superfamily members. C_LI

In pediatric hematopoietic cell transplantation (HCT) recipients, transplanted donor cells may need to function far beyond normal human lifespan. Here, we investigated the risk of clonal hematopoiesis (CH) in 144 pediatric long-term HCT survivors, compared to 115 healthy controls. CH was detected in 16% of HCT survivors, at variant allele frequencies (VAFs) of 0.01-0.31. Mutations were predominantly in DNMT3A (80%) and TET2 (20%). Older stem cell age and the HCT procedure independently increased the risk of CH (odds ratios 1.07 per year increase (p<0.001) and 2.61 for HCT (p=0.02)), indicating both aging- and transplantation-induced effects. Large clones (VAF >0.10) were found exclusively in HCT recipients. Notably, CH was also detected within 15 years after cord blood HCT. Inflammatory processes around graft infusion were associated with CH presence. Future studies are required to track the evolution of post-transplant CH and its impact on future cardiovascular disease, second malignancies and overall survival. Significance statementAs the number of long-term HCT survivors continues to increase, so does the population at risk of long-term effects. We demonstrate that pediatric HCT survivors are at increased risk of clonal hematopoiesis compared to the general population. Given the young age of these recipients, our data emphasize the need for prospective studies to assess the potential health consequences of post-transplant CH.