Existing treatments for lower-risk myelodysplastic syndromes/neoplasms (LR-MDS) focus on symptom relief. Until recently, altering the disease course was rarely considered a therapeutic objective. The first-in-class, direct, competitive telomerase inhibitor, imetelstat, demonstrated significantly higher rates of red blood cell (RBC) transfusion independence (TI) versus placebo in patients with non-del(5q), RBC transfusion-dependent LR-MDS who were relapsed/refractory to or ineligible for erythropoiesis-stimulating agents in the Phase 3 IMerge study (NCT02598661). In this exploratory analysis of IMerge, patients treated with imetelstat had greater sustained reductions in variant allele frequency of multiple mutations versus placebo recipients, which was positively associated with RBC-TI duration. Subsequent analyses showed that 70% of patients with a cytogenetic response with imetelstat achieved ≥1-year RBC-TI. Additionally, higher rates of ≥1-year RBC-TI were observed in patients with maximum variant allele frequency reduction of ≥50% in SF3B1 (58% vs. 7%), TET2 (90% vs. 9%), DNMT3A (100% vs. 13%), or ASXL1 (50% vs. 0%) and patients with ≥50% bone marrow ring sideroblast reduction (46% vs. 0%) versus patients who did not. Lastly, 60% of patients with ≥1-year RBC-TI had ≥50% reduction in telomerase activity/human telomerase reverse transcriptase RNA. These results suggest that imetelstat targets clonal progenitor cells and may modify LR-MDS biology.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are blood cancers with limited treatment options and poor outcomes. The bone marrow (BM) microenvironment significantly influences disease progression; conversely, MDS/AML remodels the niche to its advantage. Indeed, AML cells utilize the Kynurenine-HTR1B-SAA1 axis to reshape the BM niche, creating a pro-inflammatory environment that supports leukemic stem cell (LSC) proliferation. Given these insights, this study aims to investigate the role of SAA1 (Serum amyloid A1) in the progression of MDS and AML and explore potential therapeutic strategies to inhibit its activity. The quantification of bone marrow plasma levels of SAA1 in MDS and AML patients showed a strong correlation with disease severity and progression. Survival analysis revealed that SAA1 levels >4.0 µg/mL were associated with significantly poorer outcomes across all IPSS-R categories, including Very Low/Low-risk groups, highlighting its prognostic value. Functional in vitro and in vivo assays demonstrated that SAA1 selectively stimulates the proliferation of leukemic cells while sparing healthy hematopoietic stem cells (HSCs). Similarly, SAA1 enhanced the clonogenic potential of ASXL1- and TET2-mutant cells, suggesting its role in promoting stem-like properties in genetically altered hematopoietic cells, driving malignancy. Single-cell RNAseq analysis revealed that treatment of primary AML cells with SAA1 induces upregulation of pro-inflammatory cytokines, including IFN-ϒ, IL-6, TNF-α, and IL-1β, chemokines CXCL1 and CXCL8, and activates NFκB signaling, pathways promoting expansion of malignant and pre-malignant clones. In addition, it upregulates the expression of several genes associated with chemoresistance. A mouse monoclonal antibody was developed against SAA1 - 18A3. This antibody significantly inhibited the proliferation of leukemic cells and reduced NFκB activity in vitro. EdU incorporation assays demonstrated that SAA1 selectively promotes the proliferation of malignant cells in both leukemic and solid tumor lines such as T-ALL, B-ALL and pancreatic. The monoclonal antibody suppressed this effect. An in vivo study in an MLL-AF9 AML mouse model validated the therapeutic potential of anti-SAA1 antibody. Mice treated with the antibody showed reduced leukemic burden, as evidenced by bioluminescence imaging, and improved survival compared to untreated controls. Treated mice also maintained body weight and exhibited reduced disease progression, demonstrating the efficacy of the monoclonal antibody in mitigating leukemic proliferation. These findings establish SAA1 as a critical driver of leukemic progression and a valuable prognostic biomarker. The development of SAA1-neutralizing antibodies offers a promising therapeutic strategy for SAA1-targeted therapies to disrupt the inflammatory niche and improve outcomes in MDS and AML. Pallavi Budgude, Brian Chernak, Marta Galan Diez, Abdullah M. Ali, Ziwei Chen, Raul Rabadan, Martin Caroll, Azra Raza, Stavroula Kousteni. Niche driven inflammatory regulation in the pathogenesis and treatment of myeloid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3783.

Despite active therapies such as hypomethylating agents (HMA) and venetoclax advanced myeloid malignancies, outcomes in relapsed/refractory (R/R) disease or secondary acute myeloid leukemia (AML) remain poor. We previously showed that combination thioguanine (6TG) and decitabine (DEC) restores therapeutic efficacy in vitro in R/R AML (O'Dwyer K et al. Blood 2010;114:2657). Building on these observations, we completed a Phase I dose-escalation study of 6TG with DEC in advanced myeloid malignancies followed by a retrospective analysis of 42 patients treated with this regimen at our institution (Cicero KI et al. Blood 2019;134:3899). In this cohort, which included patients on- and off-study, responses were seen in 33% of patients, without a significant difference between patients who had or had not received prior HMA. This suggested that combination therapy with 6TG and DEC could overcome HMA resistance. We now present updated clinical and mechanistic data from an expanded cohort, including insights into regimen-specific effects on malignant clones and immune constitution. A retrospective chart review was performed of all adult patients at our institution with advanced myeloid malignancies who received at least 1 cycle of 6TG and DEC between 2013-2025 with formal disease assessment evaluable by bone marrow biopsy or blood. The regimen utilized the maximum tolerated dose of 6TG identified in the phase I trial. Up to 2 cycles of induction with oral 6TG 80 mg/m2/day in 2 divided doses was given on Days 1-12. Decitabine 20 mg/m2 IV was given on Days 3-12. Following this, maintenance cycles consisted of 6TG on Days 1-7 and decitabine on Days 3-7 at the same doses. Treatment continued until the time of hematopoietic stem cell transplant (HSCT), disease progression, or toxicity. The primary objective of this retrospective analysis was to determine the overall response rate (ORR). Secondary objectives were to evaluate progression-free survival (PFS) and overall survival (OS) in this population. Sixty-seven patients were identified, 53% of whom were female, with a median age of 68 years (range, 23-86 years). Thirty-four patients (51%) had secondary AML, 26 (39%) had de novo AML, 5 (7%) had higher-risk MDS, and 2 (3%) had chronic myelomonocytic leukemia (CMML). By ELN2022 risk stratification among AML patients, 40 (60%), 6 (9%), 6 (9%), and 8 (12%) had adverse, intermediate, favorable, and unknown risk disease, respectively. Patients received a median of 2 cycles of therapy (range, 1-10). Eight (12%) patients achieved complete remission (CR), 7 (10.5%) obtained a CR with incomplete count recovery (CRi), and 5 (7.5%) had morphologic leukemia-free state (MLFS), for an ORR of 30% (CR+CRi+MLFS). Nine of 20 responders (45%) had prior HMA exposure; 3 of these patients had exposure to prior HMA plus venetoclax. For the responders, the median PFS was 24 weeks (range, 7-NR), while the median OS was 50 weeks (range, 7-NR). For responders achieving composite CR (CRc: CR + CRi), median PFS was 35 weeks and median OS was 54 weeks. Two notable patients, one with CMML and another with relapsed AML, were bridged to allogeneic HSCT and are currently alive and in remission over 10 years after treatment. To elucidate response mechanisms, we performed methylome-aware whole-genome long-read sequencing and Pixelgen single-cell proteomic analysis. Pre-treatment samples from non-responders showed elevated CD44 co-localized with HLA and β2-microglobulin, while responders exhibited low CD44, uniform HLA/β2M expression, and expanded effector T-cell pools, suggesting immune activation as a key therapeutic axis. Post-treatment samples revealed persistent CD44 in non-responders, while responders expressed unexpectedly high CD36 expression in the context of re-emergence of normal stem cell marker expression. CONCLUSIONS: 6TG/DEC is a clinically active regimen in high-risk and R/R AML with a 30% ORR and favorable survival outcomes compared to historical controls. Single-cell proteomic data reveal immune-modulatory mechanisms that may underlie therapeutic response, offering a novel lens into resistance biology. Taken together, this combination remains a viable therapeutic option for elderly and unfit patients with high-risk and R/R AML who cannot tolerate intensive chemotherapy. A phase II study of 6TG and decitabine-cedazuridine as an entirely oral regimen for R/R AML irrespective of molecular phenotype is in preparation.

Introduction: Limited treatment options are available for patients with red blood cell (RBC) transfusion-dependent (TD) lower-risk myelodysplastic syndromes (LR-MDS). Imetelstat (IME), a first-in-class, direct, and competitive inhibitor of telomerase activity, was approved by the United States Food and Drug Administration in June 2024 for the treatment of RBC-TD LR-MDS in patients who were relapsed or refractory to or ineligible for erythropoiesis-stimulating agents (ESA) based on the results of the pivotal IMerge trial (NCT02598661). IMerge demonstrated significant and durable efficacy of IME (n=118) versus placebo (n=60) for ≥8-week, ≥24-week, and ≥1-year RBC-transfusion independence (TI), with a generally manageable safety profile in this patient population (Platzbecker U, Santini V, et al. Lancet. 2024). This analysis pooled data from the 3 parts of the IMerge trial (phase 2, phase 3 and QTc study) to investigate the effect of prior therapies on the clinical activity of IME. Methods: IMerge was a phase 2/3 trial with a clinical QTc study that enrolled patients with heavily RBC-TD LR-MDS who were ESA-ineligible or relapsed/ refractory. IME was administered intravenously every 4 weeks at 7.1 mg/kg (equivalent to 7.5 mg/kg IME sodium). Prior lenalidomide (LEN) and prior hypomethylating agent (HMA) use were exclusion criteria in phase 3 only. In this analysis, IME-treated patients (N=226) were pooled from phase 2, phase 3, and the QTc study of IMerge and analyzed on the basis of prior treatment as follows: ± ESA, luspatercept (LUSP), LEN, and HMA. Prior treatment was not exclusive; patients may have received >1 prior therapy. Outcomes included ≥8-week and ≥24-week RBC-TI, rates of hematologic improvement-erythroid (HI-E) based on the revised International Working Group (IWG) 2018 criteria, transfusion reduction of ≥4 U/8 weeks, and a hemoglobin (Hb) rise of ≥1.5 g/dL for ≥8 weeks. Results: As previously presented, data from the IMerge phase 3 pivotal analysis demonstrated that patients treated with IME (n=118) had ≥8-week and ≥24-week RBC-TI rates of 40% and 28%, respectively, 42% met HI-E (IWG 2018) criteria, 60% had transfusion reductions of ≥4 U/8 weeks, and 34% had a Hb rise of ≥1.5 g/dL for ≥8 weeks (Platzbecker U, Santini V, et al. Lancet. 2024). In the current analysis of all IME-treated patients pooled in IMerge (N=226), 204 had prior treatment with an ESA and 22 were ineligible for ESAs; 35 had prior LUSP, 26 had prior LEN, and 22 had prior HMA treatment. Of IME-treated patients with prior ESA therapy, 40% and 28% achieved ≥8-week and ≥24-week RBC-TI, respectively, 43% met HI-E, 64% had a transfusion reduction of ≥4 U/8 weeks, and 33% had a Hb rise of ≥1.5 g/dL for ≥8 weeks. In IME-treated patients ineligible for ESA therapy, 36% and 14% achieved ≥8-week and ≥24-week RBC-TI, respectively, 41% met HI-E, 64% had a transfusion reduction of ≥4 U/8 weeks, and 2% had a Hb rise of ≥1.5 g/dL for ≥8 weeks. Of IME-treated patients who had prior treatment with LUSP, 29% and 20% achieved ≥8-week and ≥24-week RBC-TI, respectively, 26% met HI-E, 69% had a transfusion reduction of ≥4 U/8 weeks, and 29% had a Hb rise ≥1.5 g/dL for ≥8 weeks. Of IME-treated patients with prior LEN treatment, 23% and 12% achieved ≥8-week and ≥24-week RBC-TI, respectively, 31% met HI-E, 54% had a transfusion reduction of ≥4 U/8 weeks, and 19% had a Hb rise of ≥1.5 g/dL for ≥8 weeks. Of the 22 IME-treated patients who had prior treatment with HMA, 14% and 9% achieved ≥8-week and ≥24-week RBC-TI, respectively, 18% met HI-E, 50% had a transfusion reduction of ≥4 U/8 weeks, and 14% had a Hb rise of ≥1.5 g/dL for ≥8 weeks. Conclusions: Patients who were ESA-ineligible or who had prior treatment with LUSP, LEN, or HMA in IMerge experienced clinical benefit from IME treatment, though the number of patients was small. Given the evolving therapeutic landscape for LR-MDS and the limited data available on outcomes in later lines of treatment, these results have important clinical implications, suggesting that IME demonstrates clinical activity regardless of prior therapies.

Introduction: IME is a first-in-class, direct, and competitive telomerase inhibitor approved for the treatment (tx) of certain adult pts with LR-MDS with red blood cell (RBC) transfusion-dependent anemia who are relapsed or refractory to/ineligible for erythropoiesis-stimulating agents. In the pivotal Phase 3 IMerge trial (NCT02598661), a significantly higher proportion of pts on IME vs placebo achieved ≥8-wk (40% vs 15%; P<.001) and ≥24-wk (28% vs 3%; P<.001) RBC transfusion independence (TI). The most common grade 3/4 tx-emergent adverse events with IME were neutropenia (68%) and thrombocytopenia (62%), typically occurring within the first 3 tx cycles and generally manageable and reversible. This post hoc analysis explored the possible association between IME-related cytopenias and Hb increase, a measure linked to RBC-TI achievement. Methods: Pooled pts from the 3 parts of IMerge (Phase 2/3 and QTc substudy) who received 7.1 mg/kg IME active dose (equivalent to 7.5 mg/kg IME sodium) were included. The relationship between percent reduction in platelet (PLT)/neutrophil (NEUT) counts occurring within the first 2 cycles of IME tx and subsequent outcomes was analyzed, including maximum (max) Hb increase from baseline (pre-tx Hb), hematologic improvement-erythroid (HI-E) per IWG 2006 criteria (defined as Hb rise ≥1.5 g/dL lasting 8 wk), and rates of ≥8-wk and ≥24-wk RBC-TI. Cytopenia levels were dichotomized at ≥50% reduction for PLTs and ≥75% reduction for NEUTs to assess categorical correlations with HI-E and RBC-TI responses (yes/no) using Fisher exact test and t test for group differences in Hb increase. Univariate linear regression was conducted to evaluate the association between continuous percent reduction in PLT/NEUT counts and max Hb increase. Multivariate analyses were conducted via stepwise linear regression (for max Hb increase) or logistic regression (for HI-E/TI response) with dichotomized percent reduction in PLT/NEUT counts in the presence of other baseline prognostic factors. An unadjusted nominal P value from each test was reported. Results: Data cutoff dates were 10/13/2023 (Phase 2/3) and 10/13/2024 (QTc substudy); 226 pts were included. Median values at baseline were 71 y of age, pre-tx Hb 7.8 g/dL, and PLT and NEUT counts 236×109/L and 2.6×109/L, respectively. Median IME tx duration was 34 wk. Pts with ≥50% max PLT reduction within the first 2 cycles of IME tx had a significantly greater mean max Hb increase from pre-tx (2.07 g/dL [n=169] vs 1.17 g/dL [n=39]; P=.003), HI-E rate (34.2% vs 11.9%; P=.005) and ≥24-wk RBC-TI rates (32.6% vs 7.1%; P<.001), and numerically greater ≥8-wk RBC-TI rates (42.4% vs 26.2%; P=.056) vs pts with <50% PLT reduction. As a continuous variable, PLT reduction remained significantly associated with Hb increase (P=.0095; linear regression). Similarly, pts with ≥75% max NEUT reduction within the first 2 cycles of IME tx had a significantly greater mean max Hb increase from pre-tx (2.53 g/dL [n=70] vs 1.58 g/dL [n=138]; P=.011), numerically greater HI-E rate (34.6% vs 27.7%), and comparable ≥8-wk (41.0% vs 38.5%) and ≥24-wk RBC-TI rates (30.8% vs 26.4%) vs pts with <75% NEUT reduction. As a continuous variable, NEUT reduction was also significantly associated with max Hb increase (P=.012). Multivariate analyses showed that the likelihood of achieving ≥8-wk and ≥24-wk RBC-TI was significantly correlated with the max Hb increase from pre-tx, g/dL (odds ratio [OR; 95% CI], 6.47 [3.73-11.24; P<.001] and 3.87 [2.66-5.64; P<.001], respectively). Multivariate analyses further confirmed that max Hb increase, g/dL, significantly correlated with ≥75% NEUT reduction (coefficient estimate [95% CI], 0.94 [0.32-1.57; P=.003]), and the likelihood of achieving HI-E significantly correlated with ≥50% PLT reduction (OR [95% CI], 4.16 [1.54-11.23; P=.005]). Conclusions: In this post hoc analysis, pts with ≥75% NEUT or ≥50% PLT reductions in the first 2 cycles of IME tx were more likely to have greater Hb increases from pre-tx or to achieve an HI-E response. The greater Hb increase from pre-tx emerged as a main driver for achieving ≥8-wk and ≥24-wk RBC-TI responses. Collectively, these data suggest that tx-emergent cytopenias with IME may be associated with potential for clinical benefit, similar to lenalidomide in del5q MDS. Further research is needed to confirm this on-target effect of IME resulting from its activity on clonal progenitor cells and subsequent recovery in blood cell production.

Myeloid cancers such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) remain resistant to standard of care (SOC) and targeted therapies. In this study, we demonstrate that responsiveness to therapy is associated with activation of β-catenin-JAG1 in osteoblastic cells of patients treated with all-trans-retinoic acid (ATRA). ATRA suppresses β-catenin activity in patients and leukemic mice. Consequently, it inhibits the growth and survival of MDS/AML cells from patients with active β-catenin-JAG1 signaling and promotes their differentiation. This occurs independently of cytogenetics and mutational profile. ATRA also improves disease outcome in mice with no evidence of relapse and a superior safety profile to SOC. A human anti-JAG1 antibody improves efficacy in leukemic mice and patient-derived MDS/AML cells. β-catenin activation provides an explanation for the differential response to ATRA and a mechanistic biomarker for ATRA repurposing in myeloid malignancies, potentially evading relapse and extending across a broad range of cancers.

Myeloid cancers remain resistant to standard of care (SOC) and targeted therapies. An oncogenic signal from the niche, constitutive activation of β-catenin/JAG1 signaling in osteoblastic cells (osb-β-catenin), leads to myelodysplasia (MDS) and acute myeloid leukemia (AML) and occurs in ∼40% of MDS and AML patients and patients with del(5q)-associated myeloid malignancies. We show that its levels increase with disease severity and correlate with MDS to AML transformation and a worse overall survival. Searching for regimens that block osb-β-catenin activity we identified all-trans-retinoic acid (ATRA), a regimen with disparate results in MDS and AML. 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 osb-β-catenin activity 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 osb-β-catenin, as compared to 22% among patients without this pathway active. Responsiveness was observed across patients with diverse cytogenetic and mutational profiles belonging even to adverse risk groups. Improvement occurred despite a diverse ATRA dosing schedule in combination with other therapeutic agents (AZA, VEN, VPA). Indeed, whereas there was no association between response and the degree of global DNA hypomethylation or histone acetylation among patients treated in combination with AZA/VPA, we observed high association of treatment response with baseline osb-β-catenin activity (p=0.021). Moreover, first-line AZA/VEN treatment of an AML patient resulted in relapse and CR was reached only upon ATRA addition to AZA/VEN backbone. Supporting this conclusion, treatment with ATRA monotherapy of an MDS patient with high levels of active osb-β-catenin improved disease status and was associated with complete inhibition of osb-β-catenin activity and a parallel decrease in Notch signaling in the patient’s MDS cells. The patient remained transfusion independent for at least 9 years. Mechanistically, ATRA-mediated inhibition of osb-β-catenin activity suppressed cell growth and survival and promoted differentiation of MDS/AML cells solely from patients with active osb-β-catenin without toxicity in healthy cells. Confirming findings in patient samples, ATRA administration in leukemic mice suppressed osb-β-catenin signaling thus improving disease outcome and prolonging survival by at least six-fold with no evidence of relapse and a superior safety profile to SOC. These results suggest that responsiveness to ATRA may be predicted by measuring activation of β-catenin in osteoblasts. Thus, repurposing ATRA use for the treatment of this specific group of patients may significantly improve disease outcome. Ioanna Mosialou, Abdullah M. Ali, Alvaro Cuesta-Dominguez, Brian Chernak, Marta Galan-Diez, Mael Heiblig, Ellin Berman, Joseph G. Jurcic, Steven Kornblau, Guiliermo-Garcia Manero, Azra Raza, Stavroula Kousteni. A niche driven mechanism determines response to ATRA and a mutation-independent therapeutic approach forMDS and AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3076.

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.

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.

6566 Background: In the IMerge trial (NCT02598661) of RBC transfusion-dependent (TD) patients (pts) with lower-risk myelodysplastic syndromes (LR-MDS) relapsed/refractory to or ineligible for erythropoiesis stimulating agents, imetelstat showed significant efficacy vs placebo (PBO) for 8-wk, 24-wk, and 1-y TI endpoints, with neutropenia and thrombocytopenia as the most common adverse events (Platzbecker. Lancet 2024). Supportive care was given to all pts as needed, per investigator discretion. Here we report RBC-TI rates in the absence of platelet transfusions or myeloid growth factor use. Separately, RBC-TI with mean central hemoglobin (Hb) rise of ≥1.5 g/d was assessed in all pts. Methods: Pts were randomized 2:1 to receive imetelstat (n=118) 7.5 mg/kg or PBO (n=60) Q4W IV until disease progression. Primary endpoint was 8-wk RBC-TI; 24-wk RBC-TI was a key secondary endpoint. Primary analysis cutoff was Oct 2022, with Oct 2023 cutoff for 1-y RBC-TI analyses. Results: Overall, 21/118 (18%) pts in the imetelstat group and 1/60 (2%) pts in the PBO group needed platelet transfusions; 41/118 (35%) and 2/60 (3%) pts received myeloid growth factors, respectively. Significantly higher percentages of pts achieved 8-wk, 24-wk, and 1-y RBC-TI with imetelstat vs PBO in the absence of either platelet transfusions or growth factor support (Table). In a separate analysis, 8-wk, 24-wk, and 1-y RBC-TI and concurrent Hb rise of ≥1.5 g/dL with imetelstat vs PBO occurred in 28% vs 2%, 23% vs 0%, and 17% vs 0% of pts, respectively (Table). Among responders, imetelstat increased median central Hb levels compared with PBO: 3.6 g/dL vs 0.8 g/dL for 8-wk, 4.2 g/dL vs 1.1 g/dL for 24-wk, and 5.2 g/dL vs 1.7 g/dL for 1-y RBC-TI. Conclusions: Results from this subanalysis confirm that pts who achieve RBC-TI with imetelstat do so without developing severe neutropenia and thrombocytopenia (functionally defined as needing myeloid growth factors or platelet transfusions, respectively), therefore not negating the clinical benefit of the drug. Imetelstat also led to significant rise in Hb levels in RBC-TI responders, particularly long-term responders. These data further support the efficacy of imetelstat in TD pts with LR-MDS. Clinical trial information: NCT02598661 . [Table: see text]

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.

The attenuation length of the muon content in extensive air showers provides important information regarding the generation and development of air showers. This information can be used not only to improve the description of such showers but also to test fundamental models of hadronic interactions. Using data from the LHAASO-KM2A experiment, the development of the muon content in high-energy air showers was studied. The attenuation length of muon content in the air showers was measured from experimental data in the energy range from 0.3 to 30 PeV using the constant intensity cut method. By comparing the attenuation length of the muon content with predictions from high-energy hadronic interaction models (QGSJET-II-04, SIBYLL 2.3d, and EPOS-LHC), it is evident that LHAASO results are significantly shorter than those predicted by the first two models (QGSJET-II-04 and SIBYLL 2.3d) but relatively close to those predicted by the third model (EPOS-LHC). Thus, the LHAASO data favor the EPOS-LHC model over the other two models. The three interaction models confirmed an increasing trend in the attenuation length as the cosmic-ray energy increases.

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.