Aggressive Hematologic Malignancy Research Articles

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

Malignant a-to-I RNA Editing By ADAR1 Drives T-Cell Acute Lymphoblastic Leukemia Relapse Via Attenuating dsRNA Sensing

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that frequently occurs in children, adolescents, and young adults. Approximately 10-20% of T-ALL patients will experience relapse months or years following remission and will often become refractory to further treatments. The survival of relapsed/refractory patients is very poor, with an overall survival rate of less than a 25% overall survival rate. Relapsed patients often have enriched pools of leukemia initiating cells (LICs) with enhanced pro-survival and self-renewal capacity, suggesting a potential vulnerable population for effective targeted therapies with less toxicity. An emerging research topic in LIC biology is the identification of RNA modifying enzymes that are important for LIC self-renewal and survival. ADAR1 enzymes catalyze the transition of adenosine (A) to inosine (I) in precursor double-stranded RNA (dsRNA). Epitranscriptomic A-to-I RNA editing events are widespread in the cancer transcriptome and are critical for the transition from pre-leukemic cells to fully functional LICs. Compared to myeloid leukemia, the role of ADAR1 in lymphoid progenitor maintenance and malignant transformation is not well understood . A-to-I RNA editing has a wide range of effects on RNA biology including gene expression, splicing, RNA degradation and translation, and miRNA biogenesis and/or 3' UTR targeting. The best documented functional roles of ADAR1 are suppression of the interferon (IFN) response and RNA editing of self-dsRNA to prevent abnormal activation of cytosolic self-dsRNA sensing. Concurrent deletion of the cytosolic dsRNA sensors melanoma differentiation-associated protein 5 (MDA5) and protein kinase R (PKR) is able to completely rescue embryo death and reverse the IFN signatures. Whether editing of immunogenic dsRNA and suppression of aberrant dsRNA sensing pathway could enhance LIC self-renewal capacities is an important question that has not been extensively addressed. In this study, we applied bioinformatic analysis on a large cohort of T-ALL samples to examine the function of ADAR1 in the context of T-ALL LIC maintenance. We found that ADAR1 is highly expressed in ~70% of all T-ALL patients and particularly within the LIC compartment. A thorough comparison of the A-to-I RNA editing landscape between non-relapsed and relapsed T-ALL patient cohorts revealed hyper-editing is associated with both increased risk of relapse and leukemia-associated mortality. A total of 338 under-edited and 1,472 over-edited sites were found in relapsed patients compared to non-relapsed samples. However, there was very little difference in ADAR1 expression and the overall A-to-I RNA editing levels, among various molecular subtypes of T-ALL, suggesting malignant A-to-I RNA editing is a common attribute of relapsed T-ALL regardless of the genetic mutation status. We also performed functional study in a three-dimensional human thymic organoid system and a T-ALL patient-derived xenograft (PDX) model. Depletion of ADAR1 showed striking effects in LIC survival and self-renewal with 50-90% reduction in leukemia growth. Mechanistically, we revealed complex dsRNA regulatory mechanisms of ADAR1 by directing hyper-editing of immunogenic dsRNA and retains unedited nuclear dsRNA to avoid detection by the innate immune sensor MDA5. Interesting, the dependency on ADAR1-MDA5 axis various among patients depending on the cell intrinsic level of MDA5. Collectively, our results show that ADAR1 functions as a self-renewal factor that limits the sensing of endogenous dsRNA. Thus, targeting ADAR1 presents a safe and effective therapeutic strategy for eliminating T-ALL LICs.

Spatial Response to Pivekimab Sunirine (IMGN632) In Vivo in a BPDCN Model

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy, which often manifests with skin lesions in addition to lymph node, blood, and bone marrow involvement. Standard of care treatments for BPDCN patients are intense chemotherapy or tagraxofusp (Elzonris®). Although many patients initially respond to standard-of-care therapies, most will eventually relapse. Overexpression of CD123 (IL-3Rα receptor) is a hallmark of BPDCN thus making this antigen an attractive target. Pivekimab sunirine is an antibody-drug-conjugate (ADC) comprised of a humanized anti-CD123 monoclonal antibody conjugated to a potent DNA-damaging agent, FGN849 via a cleavable linker. Pivekimab sunirine was granted orphan drug and Breakthrough Therapy designation and is currently being tested for the treatment of BPDCN patients as monotherapy and, as a triplet therapy in combination with azacitidine and venetoclax for the treatment of AML patients. Preclinically, pivekimab sunirine has demonstrated potent activity against BPDCN cells (Blood 2018 132:3956) and importantly, showed a favorable safety profile and promising efficacy in BPDCN patients (Hemasphere. 2022 Jun; 6 (Suppl): 1708-1709; ASH 2021, Abstract #1284). The goal of this study was to investigate the response to pivekimab sunirine in a pre-clinical model of BPDCN and to monitor spatial response in vivo. Pivekimab sunirine efficacy was evaluated in vitro on GEN2.2, a patient-derived BPDCN cell line, for 24h, 48h, or 72h at different concentrations. We observed a concentration and time-dependent efficacy at 48h or 72h of treatment with EC50s between 4pM and 12pM and between 1.3pM and 3pM respectively. Limited cytotoxicity was observed at the 24h time point. The efficacy of pivekimab sunirine was further confirmed in vivo in the GEN2.2 BPDCN model. Pivekimab sunirine treatment with either 0.024mg/kg or 0.08mg/kg doses at QWx3 (once weekly for 3 weeks) dosing regimen was able to restrain the tumor burden and significantly enhance survival compared to the vehicle group To assess spatial response to pivekimab sunirine, we utilized a bioluminescent (BLI) GEN2.2-luc model. Briefly, NSG mice were injected with 1x10 6 GEN2.2-luc cells followed by Fc receptor blockade on Day 2 for 24h. On Day 3, mice were treated with pivekimab sunirine (0.024mg/kg; QWx3) or vehicle intravenously. Organs were harvested on day 16 for the vehicle-treated group and day 30 for the pivekimab sunirine-treated group and tumor burden was assessed using luciferin. The vehicle group showed the presence of tumor cells in the bone marrow, spleen, and lungs. These results were confirmed by HES staining. Pivekimab sunirine treated mice showed no sign of tumor cells in any of the organs in BLI evaluation. HES staining confirmed the elimination of tumor cells in the lungs and the liver while few remaining tumor cells were observed in the spleen and the bone marrow. Overall, this work enabled us to validate the efficacy of pivekimab sunirine in a BPDCN model and to monitor its spatial effect. Data showed that pivekimab sunirine treatment significantly reduced tumor burden and increased survival compared to the vehicle group. Pivekimab sunirine treatment eradicated tumor cells in the lungs and the liver, while reducing the number of tumor cells in the spleen and bone marrow. Pivekimab sunirine is a potent ADC targeting CD123 and is highly efficacious against an aggressive BPDCN cell line model. This finding reinforces the importance of its use for the treatment of BPDCN patients.

Blastic Plasmacytoid Dendritic Cell Neoplasm Descriptors and Clinicopathologic Determinants of Survival: Analysis of a Pooled Database

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy with a propensity for cutaneous involvement. This disorder has gone through many reiterations over the years, being classified initially as a blastic NK-cell lymphoma, then a subset of AML, and finally recognized as a unique myeloid neoplasm. However, due to the poor understanding and the rarity of the disease, varied treatment approaches have historically been implemented, and no optimal data or guidelines for managing BPDCN are available. Therefore, we conducted this pooled database analysis to identify the prognostic factors, clinicopathological characteristics, and therapeutic strategies that influence survival in this disease. Methods: To study the demographic characteristics, molecular and immunohistochemical signatures, therapeutic interventions, survival, and prognostic factors, we compiled a pooled database of 273 cases that fit the diagnostic criteria for BPDCN. Kaplan-Meier survival curves were constructed. Cox proportional hazards model and Log-rank tests were used to assess the influence of demographic and clinicopathologic factors on overall survival (OS). Results: A total of 273 patients with confirmed BPDCN were identified. The median age was 61.5. There was a male preponderance with M:F of 2.4. The median duration of symptoms before diagnosis was 5 months. Eighty-one percent of patients presented with skin lesions, 65% of which were disseminated. Involvement of Lymph nodes (LN), bone marrow (BM), spleen, peripheral blood, and CNS occurred in 62%, 73%, 55%, 71%, and 57%. Constitutional symptoms were reported in 13%. The median OS and DFS of the whole group were 16 and 13 months, respectively. Patients younger than 60 had better median OS (30 vs. 10 months, p < 0.0001). The involvement of LN (p = 0.03), BM (p = 0.007), spleen (p = 0.02), and peripheral blood (p = 0.01) was associated with worse OS. Furthermore, disseminated skin involvement had worse OS than focal (p = 0.001). Though CNS involvement, presence of constitutional symptoms, elevated LDH, and TdT negative status had numerically worse OS, it did not reach statistical significance. Sex did not impact OS. Compared to no treatment, non-dose-intense chemotherapy, dose-intense chemotherapy, and stem cell transplant had incrementally superior OS (3 vs. 10 vs. 22 vs. 66 months, p < 0.0001). Patients who attained CR as their best response also had a superior median OS (26 vs. 5 months, p < 0.0001). Conclusions: This study presents updated clinicopathologic data from a large, pooled cohort of patients with BPDCN. It identifies age, organ involvement, type of therapy, and quality of response to treatment as critical determinants of OS.

DHODH Inhibitor Therapy for the Treatment of T-ALL Spares the Developing Thymus

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. Furthermore, T-ALL has not benefitted from immune-mediated therapies and continues to pose an ongoing treatment challenge with an unmet therapeutic need. DHODH has been highlighted as a potential therapeutic target in many hematologic and solid tumor malignancies including AML, neuroblastoma, glioblastoma, pancreatic cancer, and breast cancer. DHODH inhibition has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. We have confirmed this sensitivity in vitro as well as in vivo in three murine models of T-ALL, including an aggressive NOTCH driven model in syngeneic recipients and two human patient derived xenografts. Given the goal of making DHODHi therapy available to young children with relapsed/refractory T-ALL, we wished to understand the ramifications of nucleotide starvation on normal T-cells and other hematopoietic progenitors. To specifically address this, we turned our attention to the effects of DHODH inhibition on the developing thymus. This compartment would be of particular interest in pediatric patients, whose thymi have not yet finished developing. We studied juvenile mice (3 weeks) whose thymi are just reaching their peak size and growth rate. These mice were treated with the small molecule DHODHi brequinar (BRQ) 50 mg/kg every 72 hours, and were paired with age and gender-matched controls at each time point of analysis. A subset of mice were analyzed following two doses of BRQ, and thymus, bone marrow, spleen, and peripheral blood were analyzed for T-cell populations, both relative and absolute numbers. Another cohort was allowed to “recover” for 2 weeks following treatment before analysis, and a third cohort was allowed a four week recovery period. We discovered that initial treatment with brequinar significantly impacts T-cells across all stages of development ( Figure 1). This is expected, as developing T-cells are rapidly proliferative (often dividing as quickly as every 6 hours) and therefore highly dependent on their supply of nucleotides for DNA replication as well as transcription. However, after the two-week and the four-week recovery periods, these differences are no longer detectable, demonstrating the resilience of the developing thymus, and demonstrating that the effects of nucleotide starvation are temporary and reversible. These data are encouraging, as this may also suggest that the developing human thymus which is similarly rapidly proliferative in nature would also recover once nucleotide starvation was removed. Based on these data, we were interested in understanding the differential sensitivity of normal thymocytes as compared to malignant T-lymphoblasts to DHODHi. The resilience shown by normal thymocytes following treatment with BRQ may be due to their ability to tolerate periods of inhibition of de novo pyrimidine synthesis by preferentially relying on extracellular nucleotide salvage, whereas the sensitivity of malignant T-cells to DHODH may be due to their lack of ability to utilize the salvage pathway. We performed experiments to understand how cells balance de novo synthesis vs extracellular salvage. First, we performed metabolite flux tracing in Jurkat cells to directly measure nucleotide salvage. Here, we followed incorporation of isotopically labeled glutamine ( 15N) and uridine ( 13C) to determine the relative contributions of synthesis vs salvage, as de novo pyrimidine synthesis initiates from glutamine and aspartate while a cell can also use membrane nucleoside transporters (SLC28A and SLC29A family of proteins) to salvage extracellular uridine. These experiments were performed in the presence and absence of BRQ. Once BRQ is added and the cell's ability to synthesize pyrimidines is shut down, the cell must rely more on extracellular salvage as shown in Figure 2. A defective or less efficient process of extracellular salvage could help to explain this cell line's unusual dependence on de novo nucleotide synthesis. Overall, we hope that these findings will pave the way for additional preclinical studies and eventually clinical trials for pediatric patients with T-cell disease.

Provisional Prognostic Score for Blastic Plasmacytoid Dendritic Cell Neoplasm

Background BPDCN is an aggressive hematologic malignancy which often involves the skin. This disorder has gone through many iterations over the years, being classified initially as a blastic NK-cell lymphoma, then a subset of AML, and finally recognized as a unique myeloid neoplasm. While BPDCN often follows an aggressive course, its outcomes vary considerably. We conducted this study to develop a provisional BPDCN prognostic score (BPS) for this rare disease. Methods We used our constructed BPDCN database, which contains retrospective data on 273 cases. Such data included demographics such as sex, age, and race. It also included disease presentation symptoms, duration of symptoms before diagnosis, site(s) of the disease, blood counts, coexisting comorbidities, disease immunohistochemical and molecular phenotype, types of treatment, and survival outcomes. Of the 273 cases, only 218 had complete survival and outcomes data, the sample chosen for this study. Cox proportional-hazards model and Log-rank tests were used to assess the influence of clinicopathologic factors on overall survival (OS). We included factors that statistically impacted OS and scored them by the impact of their hazard ratios. Results The median OS of the cohort was 16 months. The following dichotomous variables were identified as impactful prognostic factors in this cohort: Age>60 (10 vs. 30 months), lymphadenopathy (14 vs. 42 months), bone marrow involvement (14 vs. NR months), splenomegaly (10 vs. 16 months), and disseminated skin disease (13 vs. NR months). A prognostic model was devised using these variables to identify different levels of risk. Each variable was assigned a score of 1 when present, except for age>60, splenomegaly, and disseminated skin disease, that were assigned 2 points each as they had the highest hazard ratios of all the other variables. In this exploratory cohort, low risk was assigned a score of 0-2, intermediate risk a score of 3-4, and high risk a score of 5-8. This prognostic score system led to our cohort's most optimal risk discriminatory model, where low, intermediate, and high risk had a median OS of NR, 24, and 10 months, respectively (p<0.0001). Conclusion This BPS is a promising new tool for risk-stratifying patients with BPDCN. However, it still requires prospective validation.

Functional Precision Medicine Vs Genomics Vs Clinical Experience: Feasibility Results from the Multicentric, Prospective, Randomized Controlled Exalt-2 Trial

Background Aggressive hematological malignancies have a dismal prognosis in the relapsed/refractory setting representing an unmet medical need. Despite the advance of numerous novel therapeutics to clinical routine, treatment selection at advanced disease stages typically is challenging because of chemoresistance, cumulative toxicity, patient-specific factors, and a lack of evidence-based protocols in heavily pretreated patients. Precision medicine approaches promise to extend the number of candidate drugs beyond the standard of care, ultimately providing a treatment rationale in situations where evidence-based treatment options are scarce. We have previously shown that single-cell functional precision medicine (scFPM) is clinically feasible and efficacious in patients with aggressive hematological cancers who exceeded all standard therapy lines (Snijder et al. Lancet Haematology 2017, Kornauth et al, Cancer Discovery 2022). Inspired by this proof of concept, we launched a multicentric, prospective, randomized controlled trial to assess if genetic, or functional precision medicine-guided therapy may improve patient outcomes compared to best clinical experience-guided therapy (NCT04470947). Methods EXALT-2 is a multicentric, prospective, randomized controlled trial in five academic hospitals in Austria (Vienna, Graz, Innsbruck, Linz, and Salzburg). The study aims to enroll 150 patients with aggressive hematological malignancies in a relapsed/refractory setting with an overall good performance status (ECOG ≤ 1). After inclusion, a real-time tumor-containing biopsy is obtained to confirm relapse and collect viable tumor samples from each patient. Tumor cells are subjected to single-cell functional precision medicine (scFPM) and comprehensive genomic profiling (CGP) assays. Importantly, scFPM testing comprises flow cytometry- and/or imaging-based high-throughput drug screening. Depending on the randomization arm, therapy is suggested by the multicentric, multidisciplinary EXALT-2 tumor board, which consists of at least 2 hematology specialists, a pathologist, a molecular biologist, and a pharmacist. The EXALT-2 board gives a treatment recommendation either based on i) scFPM or ii) CGP results, or iii) without any precision medicine support (Physicians' Choice, PC). If precision medicine assays fail or do not yield a treatment recommendation, a cross-over between study arms is allowed. An overview of the study design is shown in Figure 1A. Results Feasibility results are available for the first 55 included patients. Of these, 54 patients (98%) underwent real-time biopsy and were randomized with an equal distribution across study arms. 42 patients (76%) received a therapy suggestion from the EXALT-2 tumor board. The majority of these patients suffered from aggressive lymphoma . Finally, 39 patients (71% of the total, 92% of patients with a board recommendation) received treatment. 10 patients were classified as early drop-outs due to death or progression within the first 28 days of board-recommended therapy according to the study protocol. 29 patients (53% of total, 69% of patients with a board recommendation) continued on study and are available for further evaluation. Precision medicine assays were technically feasible in most instances (flow cytometry-based scFPM: 80% of tests; microscopy-based scFPM: 52% of tests; CGP: 86% of tests) and identified actionable hits in 100%, 100%, and 78%, respectively. The median time to report was shorter for scFPM than CGP testing (7 vs. 19 days, p<0.0001; Figure 1B). CGP identified a median of 1 (0-5) druggable genetic targets. In 60% of the patients (12 out of 20) with complete work-up, we observed a substantial overlap between genetic and functional (within the top 10 drug hits) results. Conclusions Our results provide further evidence that either genetic-based or functional precision medicine can be efficiently and safely implemented in clinical routine. scFPM and CGP aid to identify a rationale for innovative treatment options in a heavily pretreated cohort. Treatment recommendations can be translated to clinical application in the majority of these patients.

Durable Outcomes with Manageable Safety Leading to Prolonged Survival with Tagraxofusp for Treatment-Naive Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm: Updated Results from a European Named Patient Program

Introduction:Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive orphan hematologic malignancy that mostly presents in skin, bone marrow, and blood. BPDCN is characterized by clonal expansion of plasmacytoid dendritic tumor cells that express specific markers, including CD123. Historically, median overall survival (OS) has been ~8-14 months (mos) following chemotherapy, and the only potential cures can be conferred by hematopoietic stem cell transplantation (HSCT) following successful induction. Tagraxofusp (TAG) is a first-in-class CD123-directed therapy and the only drug approved in the US and EU to treat BPDCN. In August 2019, a global Named Patient Program (NPP) was initiated in Europe to increase patient (pt) access to TAG. We present updated results, including over two years of follow-up, in treatment-naïve pts with BPDCN from the NPP to assess TAG safety and efficacy in the real-world setting. Methods:A European multi-center, non-interventional, retrospective study was conducted in adult BPDCN pts that had access to TAG via the NPP. The main inclusion criterion was a diagnosis of BPDCN. Intravenous TAG as first-line therapy was given at 12 mcg/kg once daily on days 1-5 (up to day 10 allowed) of a 21-day cycle. Hospitalization was required for the first cycle only; subsequent cycles could be administered in an outpatient setting. Training of physicians, nurses, and pharmacists on clinical management guidelines was mandatory before initiation of treatment.The main endpoints were complete response (CR) rates and incidence and grade (G) of capillary leak syndrome (CLS). Secondary outcomes included the number of pts bridged to HSCT, OS, and safety. Results: Twenty-two treatment-naïve adult pts were enrolled in the European NPP from 08/2019 to 12/2021. Median age was 68 yrs (21 - 82), and 86% were male. Sites of disease involvement at diagnosis included skin 77%, bone marrow 59%, lymph nodes 55% (9% single; 46% multiple), and spleen 36%; 10% had CNS involvement at baseline. Median time from diagnosis to initiating TAG was 1.5 mos (range, 0.4 - 9), and the median number of TAG cycles was 3 (1 - 8). At a median follow-up of 10 mos (0.2 - 25), 18 pts had at least 1 tumor assessment. The overall response rate (ORR) was 89% (95% CI, 65 - 99), including a CR rate of 67% (95% CI, 41 - 87) and a partial response (PR) rate of 22%. Median time to ORR was 21 days (11 - 74), and median time to CR was 29 days (11 - 58). Allogeneic HSCT was undertaken in 11/22 (50%) pts; of these 7 (64%) pts were in CR prior to HSCT, and 4 (36%) pts were in PR. Of the 2 pts with CNS involvement at baseline, both had CNS clearance by intrathecal chemotherapy (IC), achieved a CR in response to TAG, and were transplanted. Median OS for all pts was 20 mos (95% CI, 10 - not reached [NR]), Figure. Median OS was NR in transplanted pts (n = 11) and was 11 mos in non-transplanted pts (n=11; 95% CI, 0.2 - NR). Median duration of response (ie, time from CR/CRi to progression or death) was 8.9 mos (95% CI, 2.2 - NR). The majority of G3/4 adverse events (AEs) or serious AEs, regardless of their relationship to TAG therapy, occurred during cycle 1. Thrombocytopenia was the most frequent hematological G3/4 AE occurring in 7 pts (32%), followed by neutropenia (18%). Overall, CLS events were observed in 10 pts (ie, edema [n=9], weight gain [n=8], hypotension [n=5], and/or hypoalbuminemia [n=1]). Nine events occurred in cycle 1, and 3 pts experienced a second CLS event. The majority of CLS events were mild to moderate (69% G≤2; 31% G3). There were no G4/5 events. Most CLS events were managed with albumin supplementation (92%) and temporary changes in TAG scheduling (69%). Median duration of CLS was 8 days, and all events resolved. No patient with CNS involvement had a CLS event. As of the data cut-off, 2 pts (9%) remain on treatment with TAG. Conclusion: Pts with treatment-naïve BPDCN who received TAG achieved durable outcomes with prolonged survival, including those with known CNS involvement. With over 2 yrs of follow-up, median OS was 20 mos with TAG, which compares favorably to chemotherapy's shorter median OS of ~8-14 mos. No new safety signals were observed. CLS was manageable; the majority of events occurred in cycle 1 and were mild/moderate, with no G4/5 events. These real-world results from a European NPP are consistent with the long-term safety and efficacy results demonstrated in the pivotal TAG monotherapy study (Pemmaraju JCO 2022) and further support TAG treatment for pts with BPDCN.

Tagraxofusp Maintenance Post-Hematopoietic Stem Cell Transplantation Provides Long-Term Survival and Manageable Safety for a Patient with Blastic Plasmacytoid Dendritic Cell Neoplasm

Introduction:Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, clinically aggressive hematologic malignancy that commonly involves four major compartments: cutaneous, bone marrow/blood, lymph nodes, and central nervous system (CNS). Tagraxofusp (TAG) is a first-in-class therapy directed to CD123, a target expressed in BPDCN, and is the only drug approved to treat BPDCN in the US and EU. TAG has demonstrated high rates of durable response and bridge to allogeneic hematopoietic cell transplantation (allo-HCT)(Pemmaraju N et al NEJM 2019). Historically, patients that undergo allogeneic allo-HCT have been reported to have long-term remissions, particularly if transplanted while in complete response; however, ~50% of patients will relapse post-allo-HCT, and strategies are needed to minimize this risk. No clinical studies to date have been conducted to assess the rate of post-HCT relapse in patients who receive TAG maintenance therapy after allo-HCT. This case study reports on a patient who was successfully treated with TAG therapy after allo-HCT in a phase 2 trial (NCT04317781). Methods and Results: A 68-year-old woman with BPDCN underwent allo-HCT and was enrolled in a TAG post-allo-HCT maintenance trial at the MD Anderson Cancer Center in 2021. Therapy prior to allo-HCT included hydroxyurea and TAG + venetoclax + mini-HyperCVAD for 5 cycles. The patient was in morphologic complete remission with low positivity by flow cytometry (positive for minimal residual disease [MRD]) at the time of allo-HCT. After allo-HCT and prior to TAG maintenance therapy, the patient had persistent cytogenic abnormalities on a bone marrow next-generation sequencing (NGS) panel. The patient received TAG 9 mcg/kg for 3 days on a 28-day cycle after allo-HCT. The patient received TAG in the post-allo-HCT setting for a total of 16 cycles. At mid-treatment (post Cycle 6, prior to Cycle 7) with TAG, the disease was in complete remission, and the bone marrow NGS panel was negative for mutations (MRD negative). The patient did not experience any high-grade toxicity or capillary leak syndrome (CLS); the only toxicity observed was weight gain (Grade 0 per CTCAE), TAG was stopped for that cycle, and the event resolved. After Cycle 16, the patient withdrew consent due to the burden of traveling from another state. Bone marrow NGS testing was repeated prior to discontinuing study treatment, and the panel remained negative for MRD, with disease still in complete remission. Conclusions: This case report demonstrates a patient experiencing a clinically meaningful benefit with TAG maintenance therapy after allo-HCT. Both prolonged survival and manageable safety, in this case, highlight the feasibility of long-term TAG maintenance post-allo-HCT to control BPDCN.

Comprehensive Analysis of Structural and Social Determinants of Health Leading to Delays in Seeking Hematologic Care: Survey of an Underserved Population with Hematologic Malignancies

Background: Disparities in outcomes based on age, race, and socioeconomic status are well-established among patients with hematologic malignancies. Timely access to health care is essential for successful treatment of aggressive hematologic malignancies. Delays in receiving treatment have been linked to worse cancer outcomes in solid tumors and is presumed to lead to worse outcomes in hematologic malignancies. Memphis, owing to its geography, draws patients from Tennessee, Arkansas, and Mississippi for complex medical care. Patients at our institution frequently belong to underserved communities, including rural, impoverished, underinsured, and racial minority populations. In this exploratory study, we sought to characterize the potential structural and social barriers that patients in an underserved population with hematologic malignancies may face accessing oncologic care around the time of their cancer diagnosis. Methods: A cross-sectional survey was conducted to better understand potential barriers to accessing hematologic care in the greater Memphis area. The survey was developed through a review of existing literature on this topic (i.e., Anderson's conceptual model for healthcare utilization), as well as from the investigators' clinical experience treating cancer patients. Several components of social determinants of health (SDOH), including psychosocial, economic, health literacy, behavioral, health care perceptions, cultural, structural, environmental, clinical, and chronic stress domains, were queried in this 56-item survey. Most items were presented as closed-ended statements on a Likert scale. Response categories for analyses were collapsed into two levels (eg, “agree” for “somewhat” or “strongly”; “concerned” for “somewhat” or “very”). Descriptive statistics, including percentages, means, medians, and ranges, were used to characterize the sample. Results: A total of 41 out of 46 consented patients completed the survey (response rate: 89.1%). The median age of respondents was 56.3 years (Table 1). Most had lymphoma (48.8%), followed by acute myeloid leukemia (17.1%) and multiple myeloma (12.2%). The majority of respondents were male (53.7%) and Black or African American (46.3%). Over half of the sample reported an annual income of less than $25,000 (57.1%) and nearly a third were uninsured at the time of their cancer diagnosis (32.5%). Approximately 44% of respondents believed that medical attention to their cancer symptoms was delayed prior to their cancer diagnosis by a doctor. Of this subset, the majority attributed the delay to economic factors (64%), followed by health literacy (58%), structural (51%), healthcare perceptions (47%), behavioral (45%), psychosocial (45%), chronic stress (43%), cultural (15%) and clinical (11%) reasons (Table 2). Conclusions and Implications: To our knowledge, this is the first survey study to investigate the factors from a patient's perspective that contributed to delays in seeking care for hematologic malignancies. This pilot study establishes the feasibility of approaching an extremely underserved population about sharing their experiences accessing medical care around the time of their cancer diagnosis as well as participating in research. Along with already well-established economic, health literacy, psychosocial, and structural barriers, respondents endorsed that health care perceptions and chronic stress contributed almost equally to delays in seeking care. Cultural and clinical barriers contributed the least. Our study findings are limited by a skewed patient selection toward those who survived initial hospitalization and treatment and is not inclusive of the sickest patients in our patient population who experienced neglected cancer symptoms. A mixed-methods, focus-group based study is planned as the next step to substantiate these findings and allow us to gain a deeper understanding into the social disparities that most heavily impact this underserved population. Acknowledgement of Funding: This research was supported by the American Society of Hematology through the ASH HONORS Award.

How Induction Cycles before Remission Influence Long-Term Survival in Acute Myeloid Leukemia

Background ： Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by accumulation and expansion of immature myeloid cells in the bone marrow, peripheral blood and other tissues. Despite the progress made in mechanisms and approval of new therapeutics, the clinical outcome of AML remains suboptimal. The achievement of complete remission (CR) after induction chemotherapy is critical because the prognosis of refractory AML was especially unfavorable. However, little is known about the association between remission after first induction therapy and the overall survival (OS) in different subgroups of AML. Study Design and Methods: We retrospectively analyzed patients with newly diagnosed AML from January 2019 to August 2022. The diagnosis and treatment strategies were based on European Leukemia Net (ELN) criteria and National Comprehensive Cancer Network (NCCN) guideline. Composite complete remission (CRc) rate (CR+CR with incomplete recovery [CRi]) were assessed at the completion of induction or re-induction therapy. Minimal residual disease (MRD) was assessed using multiparameter flow cytometry with a minimum sensitivity of 0.1%. OS was measured from the day of diagnosis until death or the last follow-up date. Results : In total, 679 patients were included and the median age was 59.0 (range, 14.0-93.0) years old. There were 56.8% (386/679) of patients who achieved CRc after the induction therapy (group A), 18.1% (123/679) achieved CRc after the re-induction therapy (group B). The median follow-up period was 24.2 months. Even though there was no significant difference between group A and group B on OS, group A exhibited improved OS when censored at allogenic hematopoietic stem cell transplantation (HSCT) compared to that in group B (group A vs group B:3-year OS rates 65.7% vs 51.1%，p=0.004，Figure A). We further explored the effects of remission depth on survival. The results showed that, after induction therapy, patients achieved CR exhibited superior OS compared with that in CRi (CR vs. CRi, 3-year OS rates 69.4% vs 51.6%, p=0.006). Also, compared with these patients who failed to achieved MRD negativity within re-induction therapy, patients achieved MRD negativity within re-induction therapy showed superior OS when censored at HSCT (MRD- vs. MRD+, 3-year OS rates 64.7% vs 34.9%, p=0.040). After the first reduction therapy, patients achieved CRc but MRD positive benefit from HSCT (HSCT vs Non-HSCT, 3-year rates 83.0% vs 57.9%, p=0.017), but in patients with MRD negativity, HSCT did not improve the survival outcomes (HSCT vs Non-HSCT, 3-year rates 76.8% vs 65.7%, p=0.078). The next-generation sequencing (NGS) results were available in 520 patients. Based on the 2022 ELN risk stratification, the 3-year OS rates were 60.3%, 44.2% and 35.0% in favorable, intermediate and adverse group, respectively. Moreover, there were 43, 47, 60 patients in favorable, intermediate, and adverse subgroups underwent HSCT subsequently. In intermediate risk subgroup, patients who failed to achieve CRc after the first induction therapy, HSCT may serve as an effective intervention to prolong survival (3-year rates 100% vs 12.1%, P<0.001,). On the contrary, patients who achieved CRc in induction therapy, the overall survival were not influenced by HSCT status (3-year rates 81.5% vs 31.7%, p=0.153, Figure B). Conclusion: Our finding indicated that remission after the first cycle of induction therapy may be a prognostic factor for OS. Moreover, earlier and deeper remission may lead to improved survival. For those patients in intermediate risk group, who achieved CRc in induction therapy or achieved CRc with MRD negativity within re-induction therapy, HSCT may not influence the survival outcome.

Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress in Adult T-Cell Leukemia/Lymphoma

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a disease model to study EP300 deficiency and an EP300-selective PROTAC degrader, as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, transmission of unrepaired inherited DNA lesions in the subsequent G1-phase and accumulation of cytosolic DNA in EP300-deficient cells. However, EP300 deficient cells show a significant defect in innate immune system activation. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress, persistent genomic instability and innate immune system evasion underlie aggressive chemo-resistant tumorigenesis in humans.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

Impact of MTHFR Polymorphisms C677T and A1298C in Acute Lymphoblastic Leukemia and Burkitt Lymphoma Patients Exposed to Methotrexate and Asparaginase

Background: Asparaginase (Asp) and high-dose methotrexate (HDMTX) are pivotal drugs in the treatment of aggressive hematological malignancies as acute lymphoblastic leukemia (ALL) and Burkitt lymphoma (BL). Asp and MTX present specific drug-related toxicities; for Asp, prothrombotic alteration of the coagulation profile, hepatobiliary and pancreatic toxicities, for MTX, renal, hepatobiliary, hematologic, gastro-intestinal and neuro- toxicity. The 5,10-methylenetetrahydrofolate reductase (MTHFR) is the enzyme involved in conversion of homocysteine into methionine. MTHFR most frequent single nucleotide polymorphisms C677T and A1298C are associated with reduced enzymatic activity up to 60%, that implies hyperhomocysteinemia and increased toxicity of MTX. Numerous studies have explored the relationship between C677T and A1298C polymorphisms of MTHFR and MTX toxicity and/or outcome in children ALL, but results are controversial. Nevertheless, it is common practice to reduce MTX dosage in adult patients in homozygosis. Aims: To explore the relationship between the MTHFR C677T and A1298C polymorphisms in homozygosis and development of MTX toxicity in a cohort of adult patients; furthermore, to evaluate the relationship with thrombotic risk in patients exposed to Asp. Materials and Methods: Data from consecutive adult onco-hematologic patients for whom MTHFR C677T and A1298C polymorphism analysis was available, treated with chemotherapeutic regimens containing HDMTX and/or Asp was retrospectively collected at a single center Italian institution. Adverse events (AEs) were graded according to CTCAE 5.0. Statistical analysis was performed with R language. The impact of MTHFR polymorphisms was evaluated by mean of a multivariate analysis adjusting Odds Ratio (OR) estimates for drug dosage and disease status. Results: Data from 78 patients, 57 affected by ALL, 3 by mixed phenotype leukemia (MPAL) and 18 by BL, was retrospectively collected. Overall, 13 (16.7%) of patients were 677TT and 8 (10.3%) were 1298CC. Patients with ALL/MPAL received mostly pediatric-inspired chemotherapy regimens (containing MTX 2.5 - 5 g/m 2 and Asp 1000-2000 UI/m 2), patients with BL received modified Rituxumab (R)-BFM protocol (MTX 1.5 g/m2); age/comorbidities-adjusted dose reduction was performed according to institutional guidelines. Median age for ALL and BL patients was 37.5 (IQR 27.0-54.2) and 33.5 (26.0-49.7) years, respectively; 54/78 (69.2%) of patients were male. Patients received a median of 3 (IQR 2-6) cycles containing HDMTX and/or Asp, for a total number of 299 cycles (94 containing Asp, 184 HDMTX and 24 both); 142 (48.0%) cycles were administered for active disease (AD), at diagnosis/relapse, and 154 (52.0%) in complete remission (CR). The impact of different dosages of Asp < 1000 UI/m 2 and ≥ 1000 UI/m² and of HDMTX <1.5 g/m 2 and ≥ 1.5 g/m 2 as well as the impact of disease status (AD vs CR) on AEs was explored. Concerning Asp therapy and thrombotic risk, 13 deep vein thrombosis (DVT) were observed, but no association between 677TT nor 1298CC was appreciated. AD status was strongly related to DVT (21.4% vs 1.8%, p 0.003). Coherently with the MTHFR-independent drug's mechanism of action, the polymorphisms had no impact on the development of Asp-related toxicities. Diversely, concerning HDMTX, in multivariate analysis, patients with 677TT were more likely to experience AST increase grade ≥3 (OR 10.55, 95%CI 1.34-83.09) while a protective role versus any grade GGT increase was observed (OR 0.09, 95%CI 0.01-0.71) (Figure 1A). Patients with 1298CC exhibited a superior incidence any grade AST/ALT increase, not confirmed for grade ≥3 (Figure 1B). No significant superior toxicity was observed in terms of renal, gastro-intestinal, hematologic AEs. The impact of a MTX dose reduction >25% of expected dose was then explored in a homogeneous population of 45 front-line pediatric-regimen treated ALL patients (35 received MTX dose ≥75% and 10 < 75%) and no impact in terms of OS and RFS was appreciated. Conclusions: The analysis ofMTHFR polymorphisms in adult patients is useful in identifying patients at higher risk for transaminitis, providing a biological parameter to integrate with hepatosteatosis, liver stiffness evaluation and BMI, for a comprehensive hepatic baseline evaluation. Patients at higher risk for hepatic complications may benefit from MTX dose reduction with no impact on disease outcome.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN):Multi-Center Collaboration and Global Registry Program

Background BPDCN is a rare, aggressive hematologic malignancy derived from the precursors of plasmacytoid dendritic cells (pDC), typically presenting with primary cutaneous dissemination and involvement of bone marrow (BM) as well as extramedullary sites including central nervous system (CNS). In 2008, it was recognized by WHO as a distinct entity and is now recognized under Myeloid/Histiocytic/Dendritic neoplasms in the 2022 5th edition update of WHO Classification. Since the recognition of this unique entity, recent efforts have been put forward for greater understanding and international collaboration, however usually focused on North America and Europe(Pemmaraju at al and Pagano et al), with this current effort representing the largest known international efforts including more parts of the world, including Asia, Middle East and Africa. Objective Due to its historical rarity and heterogeneous presentation of disease, as well as differences in availability of therapies across geo-locations, at present there is no worldwide consensus on the management of BPDCN. The outcomes remain poor, and the optimal therapy of disease remains to be determined. Launched on July 1st, 2022, the aim of our global registry is through a multi-center international collaboration to build a large database of patients with BPDCN in order to generate data-driven diagnostic and treatment recommendations. Materials and Methods The registry collects retrospective and prospective data on clinical presentation, diagnostics, treatment regimens and outcomes of patients diagnosed with BPDCN after January 1st, 2010. The data is collected globally through eCRF and Excel form. The Registry is included in ClinicalTrials.gov database (NCT05430971). Results Through July 15 th, 2023, 15 centers from 11 countries (Armenia, Canada, Cyprus, Egypt, Georgia, India, Italy, Taiwan, Turkey, UK and USA) have joined the registry and another 13 centers from USA, UK, Egypt, Iraq, Canada, Netherlands, Tajikistan, Czech Republic, Guatemala, Brazil, Serbia, Germany are onboarding. 27 retrospective and 2 prospective pts are currently included in the registry. 76 % of pts are male, in keeping with historical expectation BPDCN. Median age at diagnosis was 62 years (4-97). In 90 % of patients the tumor cells were triple positive with CD4+, CD56+, CD123+ immunophenotype. 53.6% of patients presented with cutaneous manifestation. 21% of pts received the initial treatment with AML-based regimens, 66% with ALL-based and 7% with lymphoma-based regimens. 21% of pts underwent alloSCT. 35% of pts were diagnosed after 2018, only 1 patient has received tagraxofusp, a CD123-directed therapy that received FDA approval for previously untreated or relapsed/refractory BPDCN in 2018(Pemmaraju N et al NEJM 2019). 69 % of patients achieved complete response (CR), from which 90 % were treated with ALL-based regimens. 52% of patients experienced relapse. Conclusion Current analysis of a 29-patient worldwide cohort of BPDCN demonstrates a high degree of heterogeneity in treatment regimens based on a multitude of considerations including geographic, socio-economic, drug availability, and varied clinical preferences. The outcome of ALL-based treatment was superior as compared to AML-based, but relapse rate remained high. Further global collaboration is needed to collect additional data and to identify the best diagnostic and therapeutic approaches for this challenging disease.

Patient Characteristics and Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Background: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy arising from plasmacytoid dendritic cells. It commonly presents as skin lesions with or without bone marrow, lymph node, central nervous system (CNS), or systemic involvement. Limited retrospective data have shown durable remissions after allogeneic (allo) hematopoietic cell transplantation (HCT). Methods: In this retrospective single-center analysis, we evaluated outcomes of 31 BPDCN patients treated with allo-HCT between September 2000 and June 2023 at our institution. Results: Median age of our cohort was 52 years [range 16-76 y]. Patient characteristics are summarized in the attached Table. Only 6 patients (19%) had their disease confined to skin and 4 (13%) had CNS/CSF involvement at diagnosis. Four patients (13%) had prior hematologic malignancies (HM), and 9 patients (29%) had a cytogenetic abnormality at baseline. Thirteen (42%) patients, who were evaluated by a next-generation sequencing Leukemia Mutation Panel, had TET2 mutation. Median time from diagnosis to HCT was 6.2 (3.2-42.4) months. Eight (26%) patients received tagraxofusp, matched unrelated donor was the most common donor type (39%), and fludarabine + busulfan-based conditioning was used in 17 (55%) patients. Post-transplant cyclophosphamide (PTCy) was used for GVHD prophylaxis in 23 (74%) patients. Median follow up was 23.9 (0.9-119.9) months. One-hundred day and 1-year non-relapse mortality was 9.7% and 23.1%, respectively. Grade 2 acute GVHD was seen in 9 patients (29%), and no grade 3-4 GVHD has been seen so far. Limited or extensive chronic GVHD was seen in 6 (21%) of 28 evaluable patients. Only 6 of the 31 (19%) allo-HCT patients progressed after HCT. Two-year PFS and OS was 56% and 67%, respectively. Patients receiving allo-HCT in first remission had significantly better median PFS (not reached vs. 11.3 months; p=0.034) and OS (not reached vs. 12.6 months; p=0.045). Conclusions: These results demonstrate the safety and efficacy of allo-HCT in BPDCN. Patients undergoing allo-HCT in first remission had significantly better outcomes. Prospective studies are needed to better define the role of allo-HCT in BPDCN.

Idasanutlin and navitoclax induce synergistic apoptotic cell death in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

BackgroundAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.MethodsDigoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.ResultsPrimitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.ConclusionsOur data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery.53GxHmRcFt-KhVdMmSCJrsVideo

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis.MethodsBM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay.ResultsThe study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation.ConclusionsThis study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis.AaFEVfq6u-1YqH7bnQ85uaVideo