The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.

Acute myeloid leukemia (AML) often arises from myelodysplasia (MDS), a pre-leukemic condition characterized by dysplasia and ineffective hematopoiesis. Despite extensive genetic and epigenetic profiling of AML, pathogenic mechanisms of disease development from pre-leukemic states remain largely unknown. Generation of mouse models of MDS and AML allowed us to pinpoint a global decrease in nucleoporin expression which was confirmed in public patient databases. shRNA-mediated downregulation of NUPs in mouse MDS HSPCs followed by transplantation led to fully penetrant AML with blasts in blood, bone marrow (BM) and spleen of lethally irradiated recipients. Moreover, NUP downregulation in induced Pluripotent Stem Cell (iPSC) harboring MDS-relevant SRSF2P95L and ASXL1646fs*12 mutations transformed cells as evidenced by the loss of CD34 expression, extended growth in vitro, a 2.5-fold upregulation of the leukemic biomarker CD123 and the acquisition of phenotypic characteristics of AML blasts. More importantly, shNUPs-SA HSPCs were able to engraft in NSG mice, evidencing the oncogenic properties of NUPs downregulation in a humanized in vivo setting. RNAseq analysis in mice, patients and human iPSC models revealed a signature that involves proliferative and anti-apoptotic gene pathways that promote survival of clones and arrest differentiation at the very early myeloid stage. ATAC-seq performed on a cell line with NUPs knockdown identified a significant increase in chromatin accessibility in genes involved in cell cycle, proliferation and negative regulation of apoptosis. Single-cell RNA profiling of human healthy, pre-leukemic and leukemic niche identified a unique cell population termed Fibro-MSCs that is defined by expression of Pdpn. This cell type secretes a matricellular protein, Tenascin X (TNXB), whose downregulation triggers this transformative signature. We confirmed that TNXB levels are significantly decreased in the BM of AML as compared to MDS, both in our mouse models and our patient cohorts. Transcriptomic analysis performed in bone marrow biopsies from patients with low and high blast counts showed a significant downregulation of TNXB with disease progression. These results identify a biological process and the accompanying mechanistic cascade of events within it that impose leukemia development and reveal novel targets to be exploited therapeutically.

Myeloid cancers such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) remain resistant to standard of care (SOC) and targeted therapies. Here, we identify an oncogenic signal from the niche, constitutive activation of b-catenin/JAG1 signaling in osteoblastic lineage cells, as a mechanism determining response to all-trans-retinoic acid (ATRA), a regimen with disparate results in MDS and AML. B-catenin signaling in the osteoblastic lineage is activated following hypermethylation of its regulators in MDS patients; occurs in ~40% of MDS and AML patients and patients with del(5q)-associated myeloid malignancies; its levels increase with disease severity; and correlate with MDS to AML transformation and a worse overall survival. By analyzing publicly available RNA-seq data and bone marrow biopsies collected from 14 ATRA-treated patients from four different study sites we found a strong association between inhibition of b-catenin activity in osteoblasts and/or Notch signaling in AML cells with ATRA, leading to AML improvement. An overall response rate of 100% was reached in patients with active baseline osteoblastic b-catenin, including 4 patients with a complete response (CR) and 1 patient with CR without absolute neutrophil counts of more than 10^9/L (CRn). Among patients without baseline active osteoblastic b-catenin, only 2 patients out of the 9 examined achieved a CR resulting in an overall response rate of 22%. Responsiveness was observed across patients with diverse cytogenetic and mutational profiles belonging to both intermediate and adverse risk groups. Mechanistically, ATRA-mediated inhibition of osteoblastic b-catenin activity suppressed cell growth and survival and promoted differentiation of MDS/AML cells solely from patients with active b-catenin signaling. Improvement occurs despite a diverse ATRA dosing schedule in combination with other therapeutic agents (AZA, VEN, VPA). The beneficial effect in treatment outcome appears to be dependent on baseline osteoblastic b-catenin activation since, in contrast to its high association with treatment response (Fisher's exact test, p=0.021, n=14), there was no association between response and the degree of global DNA hypomethylation or histone acetylation among patients treated in combination with AZA/VPA, even though acetylation and methylation were decreased upon treatment. Moreover, prior AZA/VEN treatment of an AML patient resulted in relapse and CR was reached only upon the subsequent ATRA addition to AZA/VEN backbone. Supporting this conclusion, treatment with ATRA monotherapy of an MDS patient with high levels of osteoblasts with activated b-catenin improved disease status and was associated with complete inhibition of b-catenin activity in the patient's osteoblasts and a parallel decrease in Notch signaling in the patient's MDS cells. The patient remained transfusion independent for at least 9 years, the entire duration of follow-up. These results suggest that responsiveness to ATRA can be due to inhibition of activated b-catenin signaling in osteoblasts. Indeed, testing this hypothesis in leukemic mice with constitutive active osteoblastic b-catenin, ATRA administration inhibited b-catenin activity in osteoblasts and improved disease with no evidence of relapse and a superior safety profile to SOC. Inactivation of ATRA receptor, RARA, specifically in osteoblasts of these mice, abrogated the protective effects of ATRA and its inhibitory effect on osteoblastic b-catenin activity demonstrating that ATRA acts through osteoblasts to improve disease progression and survival. These results provide an explanation for the differential response to ATRA suggesting ATRA repurposing in osteoblastic b-catenin associated myeloid malignancies. A circulating skeletal cell population expressing activated b-catenin, reflecting b-catenin activation status in marrow-resident osteoblastic lineage cells, can serve as a mechanistic biomarker allowing patient stratification and monitoring of treatment response. They also highlight the therapeutic potential of targeting the niche that has the potential to evade relapse and overcome SOC toxicity.

Deficiency of vitamin B-12 (B-12 or cobalamin), an essential water-soluble vitamin, leads to neurological damage, which can be irreversible and anaemia, and is sometimes associated with chronic disorders such as osteoporosis and cardiovascular diseases. Clinical tests to detect B-12 deficiency lack specificity and sensitivity. Delays in detecting B-12 deficiency pose a major threat because the progressive decline in organ functions may go unnoticed until the damage is advanced or irreversible. Here, using targeted unbiased metabolomic profiling in the sera of subjects with low B-12 levels v control individuals, we set out to identify biomarker(s) of B-12 insufficiency. Metabolomic profiling identified seventy-seven metabolites, and partial least squares discriminant analysis and hierarchical clustering analysis showed a differential abundance of taurine, xanthine, hypoxanthine, chenodeoxycholic acid, neopterin and glycocholic acid in subjects with low B-12 levels. Random forest multivariate analysis identified a taurine/chenodeoxycholic acid ratio, with an AUC score of 1, to be the best biomarker to predict low B-12 levels. Mechanistic studies using a mouse model of B-12 deficiency showed that B-12 deficiency reshaped the transcriptomic and metabolomic landscape of the cell, identifying a downregulation of methionine, taurine, urea cycle and nucleotide metabolism and an upregulation of Krebs cycle. Thus, we propose taurine/chenodeoxycholic acid ratio in serum as a potential biomarker of low B(12)levels in humans and elucidate using a mouse model of cellular metabolic pathways regulated by B-12 deficiency.

Deficiency of vitamin B12 (B12), an essential water-soluble vitamin, leads to irreversible neurological damage, osteoporosis, cardiovascular diseases, and anemia. Clinical tests to detect B12 deficiency lack specificity and sensitivity. B12 deficiency is thus insidious because progressive decline in organ functions may go unnoticed until the damage is advanced or irreversible. Here, using targeted unbiased metabolomic profiling in the sera of B12-deficient versus control individuals, we set out to identify biomarker(s) of B12 deficiency. Metabolomic profiling identified 77 metabolites, and Partial least squares discriminant-analysis (PLS-DA) and hierarchical clustering analysis (HCA) showed a differential abundance in B12-deficient sera of taurine, xanthine, hypoxanthine, chenodeoxycholic acid, neopterin, and glycocholic acid. Random forest (RF) multivariate analysis identified a taurine/chenodeoxycholic acid ratio, with an AUC score of 1, to be the best biomarker to predict B12 deficiency. Mechanistically, B12 deficiency reshaped the transcriptomic and metabolomic landscape of the cell identifying a downregulation of methionine, taurine, urea cycle, and nucleotide metabolism, and an upregulation of Krebs cycle. Thus, we propose taurine/chenodeoxycholic acid ratio in serum as a potential biomarker of B12 deficiency in humans and elucidate cellular metabolic pathways regulated by B12 deficiency.

Acute myeloid leukemia (AML) often arises from myelodysplasia (MDS), a pre-leukemic condition characterized by dysplasia and ineffective hematopoiesis. Using mouse models of MDS and AML we identified a global decrease in nucleoporin (NUP) expression that was confirmed in publicly available patient databases. shRNA-mediated downregulation of NUPs in mouse MDS HSPCs and transplantation led to fully penetrant AML with blasts in blood, bone marrow (BM) and spleen. To examine the relevance of NUPs in human disease we downregulated NUPs expression in a model of induced Pluripotent Stem Cell (iPSC)-derived HSPCs harboring the MDS-relevant SRSF2P95L and ASXL1646fs*12 mutations (SA). shNUPS-SA HSPCs overcame exhaustion and loss of CD34 expression, typical of MDS cells. Transformed cells maintained growth for as long as 10 months, acquired phenotypic characteristics of AML blasts and presented a 2.5-fold upregulation of the leukemic biomarker CD123. shNUPS-SA HSPCs engrafted in NSG mice, establishing the transformative potential of NUP downregulation in a humanized in vivo model. RNAseq analysis of HSPCs from mouse and human models of AML versus MDS and patient samples revealed upregulation of genes promoting epithelial-to-mesenchymal transition (EMT). Master regulator analysis between patient-derived MDS and AML HSPCs and their stroma identified the secreted matricellular protein Tenascin X as a candidate regulator of NUPs expression. TNXB levels decreased in BM plasma of AML as compared to MDS patients and in the BM of AML as compared to MDS mice. Mass spectrometry analysis identified the presence of a TNXB protein mainly consisting of the fibrinogen-like domain linked to active TGF-β-mediated activation of EMT. The identification of EMT as a signature of transformation in a non-solid cancer uncovers a novel pathway of AML invasiveness that could be potentially targetable.

Metastatic cancer is a leading cause of death in cancer patients worldwide. While circulating hybrid cells (CHCs) are implicated in metastatic spread, studies documenting their tissue origin remain sparse, with limited candidate approaches using one–two markers. Utilizing high-throughput single-cell and spatial transcriptomics, we identified tumor hybrid cells (THCs) co-expressing epithelial and macrophage markers and expressing a distinct transcriptome. Rarely, normal tissue showed these cells (NHCs), but their transcriptome was easily distinguishable from THCs. THCs with unique transcriptomes were observed in breast and colon cancers, suggesting this to be a generalizable phenomenon across cancer types. This study establishes a framework for HC identification in large datasets, providing compelling evidence for their tissue residence and offering comprehensive transcriptomic characterization. Furthermore, it sheds light on their differential function and identifies pathways that could explain their newly acquired invasive capabilities. THCs should be considered as potential therapeutic targets.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

In haematological malignancies, dysplastic hematopoietic stem and progenitor cells (HSPCs) can remodel bone marrow mesenchymal stem cells (BMSC) in their favour. However, it is unknown whether BMSCs remodelling occurs during transformation of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Transcriptomic analysis and exome sequencing of BMSCs from bone biopsies of MDS patients before and after AML transformation, showed metabolic dysregulations at the mitochondrial level during transformation. Specifically, BMSCs from AML-transformed patients showed downregulation of mitophagy genes and upregulation of genes encoding mitochondrial proteins. Mitochondrial numbers increased but presented compromised metabolic function as evidenced by MitoStress test and prevalence of donut-shaped mitochondria. Interestingly, expression of mitochondrial trafficking genes was increased in AML as compared to MDS BMSCs. As a result, MSCs and dysplastic HSPCs exchange mitochondria via Tunneling Nanotubes (TNTs). This exchange occurs so that AML cells transfer dysfunctional mitochondria to BMSCs, whereas BMCSs transfer functional mitochondria to AML blasts. Mitochondrial transfer reduces the number of CD45+ AML cells indicating that it alters their profile to a more immature population. Notably, these events, do not occur among healthy BMSCs and HSPCs. Further, inhibition of TNTs formation using Jasplakinolide, reduced mitochondrial transfer and the number of dsRed+ MLL-AF9 cells in the bone marrow and spleen of MLL-injected mice, and maintained tissues architecture. As a resulted, it slowed disease progression and prolonged survival in leukemic mice. These observations demonstrate that BMSCs provide functional mitochondria to MDS cells. The transfer allows malignant cells to overcome their metabolic stress and favour their hyperproliferation as AML blasts. In haematological malignancies, dysplastic hematopoietic stem and progenitor cells (HSPCs) can remodel bone marrow mesenchymal stem cells (BMSC) in their favour. However, it is unknown whether BMSCs remodelling occurs during transformation of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Transcriptomic analysis and exome sequencing of BMSCs from bone biopsies of MDS patients before and after AML transformation, showed metabolic dysregulations at the mitochondrial level during transformation. Specifically, BMSCs from AML-transformed patients showed downregulation of mitophagy genes and upregulation of genes encoding mitochondrial proteins. Mitochondrial numbers increased but presented compromised metabolic function as evidenced by MitoStress test and prevalence of donut-shaped mitochondria. Interestingly, expression of mitochondrial trafficking genes was increased in AML as compared to MDS BMSCs. As a result, MSCs and dysplastic HSPCs exchange mitochondria via Tunneling Nanotubes (TNTs). This exchange occurs so that AML cells transfer dysfunctional mitochondria to BMSCs, whereas BMCSs transfer functional mitochondria to AML blasts. Mitochondrial transfer reduces the number of CD45+ AML cells indicating that it alters their profile to a more immature population. Notably, these events, do not occur among healthy BMSCs and HSPCs. Further, inhibition of TNTs formation using Jasplakinolide, reduced mitochondrial transfer and the number of dsRed+ MLL-AF9 cells in the bone marrow and spleen of MLL-injected mice, and maintained tissues architecture. As a resulted, it slowed disease progression and prolonged survival in leukemic mice. These observations demonstrate that BMSCs provide functional mitochondria to MDS cells. The transfer allows malignant cells to overcome their metabolic stress and favour their hyperproliferation as AML blasts.

7058 Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid malignancies associated with a myriad of deleterious outcomes and a 5-year relative survival of 37%. MDS risk stratification is key for optimal treatment decisions. DNA methylation is associated with MDS biology due to frequent somatic mutations in genes (i.e. TET2, DNMT3A, IDH1, IDH2, and WT1) that affect DNA methylation. We hypothesized that methylation-based markers may help stratify risk and improve IPSS-R (the Revised International Prognostic Scoring System). Here, we evaluated the potential of both Bone Marrow (BM) Whole-genome Bisulfite Sequencing (WGBS) and Serum-based Targeted Methylation (TM) Sequencing to predict survival of MDS patients. Methods: We conducted paired BM WGBS and Serum-based TM sequencing on a cohort of 127 patients with MDS (N = 104) and secondary AML (N = 23) treated at the Columbia University Medical Center. We categorized patients based on an overall survival of > 3 (“long”, N = 50) or < 3 years (“short”, N = 77). We then identified differentially methylated regions (DMRs) differing between long and short survivor groups, and analyzed the biological pathways and functions enriched in these regions. To assess survival association, we trained a random forest classifier on principal components (“methylation classifier”) on the methylation fractions from a subset of 96 Serum and 98 BM MDS patient samples to predict long vs. short survival, and compared this against a logistic regression IPSS-R model. We optimized the hyperparameters and assessed classifier performance using six-fold nested cross-validation. Finally, we conducted a multivariable cox regression with predicted scores from the methylation classifier, IPSS-R scores (recalculated at sample collection), age, and gender. Results: We identified a total of 7,742 DMRs in the BM WGBS and 14,093 DMRs in the Serum TM as significantly different between long and short survivor groups. Signaling pathways for calcium, cAMP, MAPK, Rap1, and PI3K-Akt were significantly enriched in DMRs. We also identified a previously-reported CpG island hypermethylation signature associated with AML progression in BM and Serum. The methylation classifier achieved a comparable AUROC compared to IPSS-R logistic regression for both BM WGBS (0.80 vs 0.78) and Serum TM (0.77 vs 0.73) samples. Multivariable Cox regression demonstrated that both predicted scores from the methylation classifier (Serum p =0.002; BM p =0.016) and IPSS-R scores (Serum p =0.003; BM p =0.004) are significant predictors of survival. Conclusions: We demonstrated methylation-based sequencing represents a promising new tool for MDS patient risk stratification. As IPSS-R relies on bone marrow aspirates, the Serum-based TM assay offers a less-invasive method for MDS risk stratification.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 ( Grem1 ) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1 -lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1 -lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1 -lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p<0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

<p>PDF file - 407K, DNA-damage-induced phosphorylation of FANCM is ATR dependent (S1); Peptides released from FANCM protein were analyzed for putative phosphorylation sites by mass spectrometry (S2); Immunoblot showing that antibody raised against phosphorylated S1045 specifically recognizes the ectopically expressed wildtype, but not S1045A mutant, form of FANCM (S3); UV mediated DNA-damage-induced phosphorylation of FANCM at S1045 (S4); Phosphorylation at S1045 is not required for FANCM binding to the FA core complex but for FANCD2 monoubiquitination (S5); MMC-induced chromatin association of the FA-core complex is impaired in S1045A-expressing cells (S6); Recruitment of FANCM to ICL site is ATR dependent (S7); S1045A mutant-expressing cells do not show any gross change in cell-cycle distribution (S8); Expression of FLAG tagged FANCMK117R in the context of knockdown of the endogenous protein (S9).</p>

Cells of the surrounding bone marrow microenvironment (niche) have emerged as important regulators of myeloid disease development and progression, leading to myeloproliferative neoplasms, myelodysplasia (MDS) or acute myeloid leukemia (AML). This not only highlights the complexity of the disease but may, at least in part, explain the limitations of current malignant cell targeted therapies to prevent relapse and at the same time opens new avenues for therapeutic intervention. To test this hypothesis, we examined here the therapeutic potential of targeting a potent, niche-driven oncogenic pathway, constitutive activation of b-catenin/Jagged1 signaling in osteoblasts. In humans, this pathway is activated in approximately 40% of MDS and AML patients; and also following hypermethylation of its regulators in MDS patients. Its activation levels increase with disease severity, correlate with MDS to AML transformation and with del(5q)-associated myeloid malignancies. In mice, it leads to MDS rapidly progressing to AML. To test its therapeutic potential, we inhibited Jagged1. We generated a chimeric human-mouse neutralizing antibody that efficiently and specifically binds JAG1 (anti-JAG1) and inhibits Notch1-induced signaling. Administration of anti-JAG1 in leukemic mice with activated b-catenin/Jag1 in their osteoblasts rescued anemia, thrombocytopenia, neutrophilia and lymphocytopenia, relieved myeloid differentiation block and eliminated blasts. Body weight increased with time and lethality was abrogated in treated mice. Blood chemistry profiling indicated lack of any toxicity following treatment as indicated by normal liver and kidney function and absence of inflammation, dyslipidemia or pancreatitis. In contrast, chemotherapy at a dose simulating the induction regimen used in patients, dramatically exacerbated anemia, thrombocytopenia and lymphocytopenia without decreasing blasts leading to increased lethality due to bone marrow failure. Emphasizing relevance to human disease, anti-JAG1 treatment of patient-derived samples with activated b-catenin/JAG1 in their osteoblasts, inhibited MDS and AML cell growth and survival and promoted myeloid and erythroid differentiation through its actions on osteoblasts. Responsiveness was observed across patients belonging to diverse disease subtypes and categories including patients with adverse cytogenetics and high-risk groups. Confirming the specificity of anti-JAG1 action, no effect was observed in cells from patients without activated b-catenin/JAG1 in their osteoblasts or healthy subjects and the magnitude of the response correlated with the levels of b-catenin/JAG1 activation in osteoblasts. These results suggest the therapeutic efficacy of blocking JAG1 and its superiority to chemotherapy in osteoblastic, b-catenin-driven MDS/AML that could impact 1/3 of MDS and AML patients. In addition, they suggest that targeting the niche may be an approach to avoid toxicity and overcome MDS/AML cell mutation dependence and clonal resistance that follows standard of care, and therefore, prevent relapse. Citation Format: Ioanna Mosialou, Abdullah M Ali, Rachel Adams, Adam Corper, Catherine M Woods, Xiaomin Fan, Azra Raza, Stavroula Kousteni. A niche directed therapy for the treatment of myelodysplasia and acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A25.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo , thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro , dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies. Graphical abstract: