High-risk Myeloid Malignancies Research Articles

Smart Conditioning with Venetoclax-Enhanced Sequential FLAMSA + RIC in Patients with High-Risk Myeloid Malignancies

Up to 50% of patients with high-risk myeloid malignancies die of relapse after allogeneic stem cell transplantation. Current sequential conditioning regimens like the FLAMSA protocol combine intensive induction therapy with TBI or alkylators. Venetoclax has synergistic effects to chemotherapy. In a retrospective survey among German transplant centers, we identified 61 patients with myeloid malignancies that had received FLAMSA-based sequential conditioning with venetoclax between 2018 and 2022 as an individualized treatment approach. Sixty patients (98%) had active disease at transplant and 74% had genetic high-risk features. Patients received allografts from matched unrelated, matched related, or mismatched donors. Tumor lysis syndrome occurred in two patients but no significant non-hematologic toxicity related to venetoclax was observed. On day +30, 55 patients (90%) were in complete remission. Acute GvHD II°–IV° occurred in 17 (28%) and moderate/severe chronic GvHD in 7 patients (12%). Event-free survival and overall survival were 64% and 80% at 1 year as well as 57% and 75% at 2 years, respectively. The off-label combination of sequential FLAMSA-RIC with venetoclax appears to be safe and highly effective. To further validate these insights and enhance the idea of smart conditioning, a controlled prospective clinical trial was initiated in July 2023.

Treosulfan-Based Reduced-Toxicity Conditioning for Elderly Patients with High-Risk Myelodysplastic and Myeloproliferative Disease for Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Allogeneic hematopoietic stem cell transplantation (alloHCT) is the only potential curative treatment for patients (pts) with high-risk myeloid malignancies. Despite advances in the supportive care over the past decades, alloHCT is still associated with significant toxicity, especially in the elderly population. The choice of an adequate conditioning regimen has become essential in order to prevent treatment-related morbidity and mortality. Since myeloablative conditioning (MAC) is mostly restricted to the younger population due to the high toxicity, reduced-intensity conditioning (RIC) provides an alternative solution. However, especially for pts with high-risk disease, a potential increase in the incidence of relapse must be taken into account. The advent of reduced-toxicity conditioning regimens (RTC), mostly based on treosulfan in combination with fludarabine, offers a balance between a favorable toxicity profile and a strong anti-neoplastic effect. Considering the increasing rates of alloHCT among older pts in the past decades, finding the optimal conditioning regimen is still an unmet need for this collective. The objective of this study is to analyze the outcome of pts aged ≥60 years who underwent alloHCT for myelodysplastic or myeloproliferative disease and received a treosulfan-based conditioning. In order to maximize the anti-leukemic effect of the conditioning in this population we chose the myeloablative dose of treosulfan of 14g/m2 daily. Materials and methods We retrospectively analyzed the outcome of adult pts ≥60 years with myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) or myelofibrosis (MF) who underwent their first allo-HCT from a matched or mismatched donor over a period of 10 years at the adult Bone Marrow Transplantation Unit of our institution. The conditioning consisted of a combination of treosulfan 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -2, alone or combined with 2 Gy TBI (day 0) for MDS and treosulfan, 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -3 for CMML and MF. Endpoints of this analysis were engraftment rate (together with time to engraftment), relapse-free survival (RFS), overall survival (OS), and non-relapse mortality (NRM). Continuous variables were illustrated as median values. NRM, RFS and OS for the whole cohort were analyzed with the Kaplan-Meier method and presented as survival functions. Results Between 01/2012 and 12/2022 91 consecutive elderly pts were eligible for this analysis. Patient and transplant characteristics are listed in Table 1. The median follow-up after allo-HCT was 3.12 years (4 months-10.5 years). Median age was 65 years, 76% (n = 69) of patients had an EBMT (European Society for Blood and Marrow Transplantation) risk score of ≥ 4. In 20 of 91 pts (22%) a 9/10 matched unrelated donor was selected, due to the lack of a more suitable donor. The standard graft versus host disease (GVHD) prophylaxis was a combination of calcineurin inhibitors (CNI) +/- mycophenolate mofetil and Anti-T-lymphocyte globulin (ATG) (n= 85, 94%). Neutrophil engraftment was achieved in 98% (n= 89) of pts. The median time to engraftment was 13 days (12, 15). Fourteen of 91 pts (15%) developed severe (grade III-IV) acute GVHD, and 44% (n= 40) had chronic GVHD of any grade. Considering early toxicity, 2 patients of 91 (2%) died before day +30 after alloHCT due to transplant-related toxicity, overall 6 of 91 (7%) in the first 100 days after alloHCT. The 2-year OS was 55% (95% CI, 44.7-.66.2) (Figure 1) and RFS was 48.5% (95% CI, 38.9-60.5). The 2-year NRM was 37% (95% CI, 33.5-39.5). No significant differences have been observed in the outcome of the different disease entities. Main causes of death were infections (n= 15, 32%), GVHD (n=13, 27.7%) and disease relapse (n= 13, 27.7%). Conclusions Our retrospective analysis suggests that a treosulfan-based RTC with myeloablative potential in an elderly and frail population of pts with high-risk myeloid diseases is feasible, with an excellent engraftment rate and a limited early toxicity, and promotes disease control. We report a 2-year NRM of 37%, which was partially due to severe GvHD in a cohort of pts with a relatively high-proportion of mismatched donors (20%). The role of post-transplant cyclophosphamide as backbone of the GvHD prophylaxis of RTC regimes should be further evaluated, in prospective trials.

CUX1 Levels Control Hematopoietic Stem Cell Fate and Plasticity

Hematopoietic stem cells (HSCs) sustain lifelong, multipotent output, however, the molecular mechanisms governing HSC homeostasis remain unclear. Elucidating this process is key to understanding the imbalanced differentiation and self-renewal evident in myeloid malignancies. CUX1 is a transcription factor that is monoallelically mutated or deleted in high-risk myeloid malignancies. We previously reported that CUX1 has critical, dose-dependent roles in hematopoiesis and myeloid neoplasia. The haploinsufficient nature of CUX1 in myeloid neoplasia and the broad dose-dependent requirement of CUX1 in hematopoiesis led us investigate the role of CUX1 in earliest stages of hematopoiesis. We generated a CUX1 reporter mouse in which mCherry is fused in-frame with the C-terminus of CUX1 to study how the level of CUX1 in HSCs affects their behavior. Overall, we find that CUX1 protein levels within the HSC compartment delineates stem cell activity. Stringently defined immunophenotypic HSCs with low CUX1 levels (CUX1 Dim) are phenotypically dormant, as assessed by transcriptional profiling and proliferation assays, as opposed to CUX1 Bright HSCs which have greater cell cycle participation. In transplantation assays, CUX1 Dim HSCs are initially platelet-biased but give rise to long-term, multilineage reconstitution. CUX1 Bright HSCs are initially erythroid-biased but lack long-term HSC activity and the capacity for self-renewal. In other words, despite immunophenotypic HSC markers, CUX1 Bright HSCs are not, by definition, HSCs. By most metrics, CUX1 Intermediate HSCs exhibit a distinct, albeit intermediate, phenotype. Therefore, along a continuous gradient of CUX1 expression, HSCs gradually lose the ability to self-renew as CUX1 expression increases. Remarkably, knockdown of CUX1 in CUX1 Bright HSCs re-endows these cells with the capacity for long-term multilineage chimerism, suggesting low levels of CUX1 are required for stem cell activity in HSCs. Inversely, overexpression of CUX1 in HSCs promotes their expansion and ultimate exhaustion, further demonstrating that the level of CUX1 controls HSC fate. Altogether, we report that low CUX1 levels are required for HSC self-renewal and implicate CUX1 as a molecular rheostat for HSC behaviors such as cell division and differentiation. Further, we describe an unprecedented capacity for cells without demonstrated HSC activity to acquire HSC capacity and a novel role for CUX1 in preventing this plasticity.

Low-Dose Decitabine Plus Venetoclax As Post-Transplant Maintenance for High-Risk Myeloid Malignancies

Background: Allogeneic stem cell transplantation (allo-SCT) is potentially curative for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, post-SCT relapse remains a major cause of treatment failure for these patients and strategies to prevent post-transplant relapse are urgently needed. The combination of venetoclax (VEN) with either azacitidine (AZA) or decitabine (DEC) has shown promising results in older or unfit patients with AML and is now being investigated in younger patients as well. Herein, we report our experience of administering low-dose DEC and VEN as post-transplant maintenance in patients with high-risk myeloid malignancies. Methods: A retrospective chart review was performed to evaluate outcomes of patients with AML and MDS that received post-SCT maintenance with DEC and VEN at the University of Alabama at Birmingham. DEC was administered at 10mg/m 2 on days 1-3 per cycle and VEN was given at a target dose of 200mg on days 1-28 for AML and days 1-14 for MDS. The dose of VEN was reduced to 20mg when administered with voriconazole or posaconazole or 100mg when administered with fluconazole or isavuconazium. The cycle length was 4 weeks. Results: We identified 19 patients between March 2021 and May 2023 that received post-SCT DEC/VEN for AML and MDS (Table 1). The median follow-up was 10m (range 3-26m). The median age was 60y (range 34-69y). There were 10 (53%) females and 13 (68%) non-Hispanic whites (NHWs). Fourteen patients had AML and 5 had MDS. For patients with AML, 10 had poor-risk and 4 had intermediate-risk disease per ELN 2017. At the time of transplant, 11 patients with AML were in CR1 (CR1=6, CRi1=5), 2 were in CR2 (CR2=1, CRi2=1) and 1 was in CR3. Three patients with AML had MRD+ at the time of transplant (CRi1=2, CR3=1). For patients with MDS, 2 had very-high and 2 had high-risk disease per R-IPSS. One patient had therapy-related MDS. Two patients with MDS had CR, 2 had stable disease and 1 had hematological-improvement at the time of transplant. Overall, 6 patients (32%) had a TP53 mutation. Donor type was matched-related in 5 patients, matched-unrelated in 12 patients and haploidentical in 2 patients. Seventeen patients received reduced-intensity conditioning (RIC). GVHD prevention consisted of tacrolimus and methotrexate in 14 patients and post-transplant cyclophosphamide-based in 5 patients. The median time to initiating maintenance therapy was 69d (range 54-200d) post-SCT. The median number of cycles administered was 6 (range 2-24). Grade III-IV neutropenia, anemia and thrombocytopenia occurred in 5, 1 and 2 patients, respectively. Two patients had febrile neutropenia and one patient had candida esophagitis. Five patients required a reduction or interruption in the dose due to adverse events. In all cases, the VEN duration was shortened by 1 week. The cumulative incidence (CI) of day 100 grade II-IV and III-IV aGVHD was 13% and 5%, respectively. There were no grade IV cases. The 1y CI of moderate-severe cGVHD was 26%. To date, one patient who received a RIC haplo-SCT for poor-risk AML in CR2 relapsed 14m after SCT. The 1y non-relapse mortality was 11%. The 1y OS for the entire cohort was 84% (Figure 1). Conclusion: Our preliminary data suggests that the combination of low-dose DEC plus VEN is a safe and potentially effective strategy to reduce the risk of post-SCT relapse. Adverse effects mainly included manageable cytopenias with no increase in risk of GVHD. The efficacy of the combination appears to be promising but needs longer follow-up as well as confirmation in large, randomized studies.

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

A Phase 2, Single-Arm, Multicohort, Open Label, Multicenter Trial of Off-the-Shelf Natural Killer Cells (SAR445419) in Patients with High-Risk Myeloid Malignancies Undergoing Allogeneic HSCT

Background and Significance: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the risk depends on parameters such as age and conditioning intensity, relapse is experienced in 30-50% of patients after conventional HSCT in high-risk AML/MDS. Initial safety and post-HSCT relapse risk reduction results were reported in a phase I study of haploidentical (haplo) donor-derived natural killer (NK) cells expanded ex vivo on feeder cells expressing IL-21 and 4-1BBL in conjunction with haplo-HSCT (Ciurea et al, Leukemia, 2022). This study supported exploring SAR445419 (SAR'419), an off-the-shelf (OTS) product, derived from universal donor NK cells and expanded ex vivo in a feeder cell-free system using plasma membrane particles bearing membrane-bound IL-21 and 4-1BBL. Here, we aim to test whether SAR'419 can mediate an effective anti-leukemia response and reduce post-transplant relapse rate when administered in the peri-transplant period. Study Design and Methods: In this multicenter, multicohort, phase 2, single-arm trial (NCT05726682), patients with high-risk AML/MDS planned for allo-HSCT from matched sibling, matched unrelated or haplo donors will be enrolled in 2 cohorts. Cohort A will comprise patients aged 18‒65 years undergoing myeloablative conditioning with hematopoietic cell transplantation comorbidity index ≤3 and cohort B will comprise patients aged 18‒75 years undergoing reduced intensity conditioning. Patients with prior allogeneic transplantation or AML beyond first complete remission will be excluded from the study. Patients will undergo HSCT conditioning starting on Day ‒7 and will receive a peripheral blood stem cell graft on Day 0. Prophylaxis for Graft-versus-host disease (GVHD) using cyclophosphamide, mycophenolate mofetil, and tacrolimus will start on Day +3. SAR'419 will be administered as adjunctive therapy to the HSCT on Day ‒2, Day +7, and Day +28. To confirm the anticipated safety profile of SAR'419, a safety run-in will be conducted in 6‒12 patients in each cohort prior to open enrollment. Patients will be enrolled until 51 and 44 patients in cohorts A and B, respectively, receive at least the first 2 doses of SAR'419 at the target dose confirmed during safety run-in. In total, approximately 107 patients will be enrolled. The primary endpoint, relapse free survival (RFS), will be assessed at 12 months post-HSCT. Additional primary endpoints include frequency of graft failure, life-threatening infusion-related reactions or cytokine release syndrome, tumor lysis syndrome, immune cell-associated neurotoxicity syndrome, acute GVHD, overall mortality at 100 days post-HSCT, non-relapse mortality, and cytomegalovirus reactivation/infection. Secondary endpoints are safety, tolerability, and the impact of SAR'419 on HSCT outcomes, stem cell engraftment, GVHD, infection, and quality of life. Exploratory assessments include evaluation of the impact of SAR'419 on immune reconstitution, cytomegalovirus seroconversion, minimal residual disease, resource utilization, SAR'419 immunogenicity and persistence and their impact on outcomes in whole blood (including serum and plasma) and bone marrow aspirates. RFS will be reported as the percentage of patients who are relapse free and alive at 12 months from the date of HSCT and who received at least the first 2 planned doses of SAR'419. Adverse events will be graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v5.0 and will be summarized using descriptive statistics.

Optimized Double-Unit Cord Blood Transplantation Mitigates Transplant-Related Mortality Resulting in High Progression-Free Survival in Adults with Hematologic Malignancies

Background : Double-unit cord blood transplantation (dCBT) with intermediate intensity conditioning (Cy50/ Flu150/ Thio10/ TBI400cGy) and cyclosporine-A (CSA)/ mycophenolate mofetil (MMF) graft-versus-host disease (GVHD) prophylaxis has been associated with high progression-free survival (PFS) in adult patients (pts) with hematologic malignancies ( Barker JN et al, Blood Advances 2020). Now, our Center has investigated multiple refinements to further mitigate transplant-related mortality (TRM). Herein, we have analyzed transplant outcomes in these adult dCBT pts to establish an optimized dCBT platform. Methods : Consecutive adult first allograft pts with high-risk hematologic malignancies who underwent first allografts with intermediate intensity dCBT between 3/2018-8/2022 were analyzed. Optimizations involved further refined unit selection placing highest priority on unit quality (i.e. optimized banking practices) & CD34+ cell dose over HLA-match, ensuring therapeutic (CSA) levels (> 250 ng/mL) by day 0, prompt treatment of pre-engraftment syndrome (PES) & letermovir prophylaxis in CMV seropositive pts from day +7. Also, we have investigated 2 strategies to mitigate acute graft-versus-host disease (aGVHD): pts early in the study period received CSA, MMF & tocilizumab (8 mg/kg on day -1) prophylaxis ( Toci Group), whereas recent pts ( Recent Group) received CSA/ MMF with prompt therapy of clinically diagnosed aGVHD. Results : Of 74 patients (Table), 49 were in the Toci Group, & 25 in the Recent Group. Most pts were transplanted for acute leukemia (n = 55, 74%) & over half (n = 39, 53%) had non-European ancestry. Distribution of diagnoses, CMV serostatus, HCT-CI & graft characteristics were similar between the groups but Recent Group pts tended to be younger (38 vs. 47 years, p = 0.071). Toci Group pts had delayed neutrophil recovery [engraftment incidence 94% (95%CI: 87-99) at a median of 25 days (range 16-40), one graft failure], but low rates of PES (n = 19, 39%). The day-100 incidence of grade II-IV & III-IV aGVHD were 69% (95%CI: 56-82) & 10% (95%CI: 2-19), respectively. One pt (false negative CMV-serostatus) who did not receive letermovir had CMV infection. One-year TRM was 16% (95%CI: 6-27) with a 1-year relapse rate of 6% (95%CI: 3-13). TRM was due to infection (n = 3), aGVHD (n = 3, 1 with concurrent COVID-19 infection), multiorgan failure (n = 2) & graft failure (n = 1). With a median follow-up of 47 months (range 28-62), the 1-year OS was 84% (95%CI: 73-94) & 1-year PFS was 78% (95%CI: 66-89). By contrast, the engraftment incidence in Recent Group pts was 100% with a faster median neutrophil recovery of 19 days (range 11-36, p = 0.002) & all engrafted platelets at a median of 36 days (range 24-92). However, most Recent group pts developed PES [n = 20 (80%), p < 0.001] although this responded promptly to short course corticosteroids. Day 100 grade II-IV aGVHD was also higher [92% (95%CI: 81-99), p = 0.009], although grade III-IV aGVHD was low [8% (95%CI: <1-19)] & similar to tocilizumab pts. Day 100 incidence of CMV was 0%. With a median follow-up of 17 months (range 6-50), the 1-year TRM in Recent Group pts is 0% with a 1-year relapse incidence of 13% (95%CI: 7-27). Consequently, the 1-year OS & PFS are high at 96% (95%: 89-99) & 87% (95%CI: 74-99), respectively (Figure). Conclusions: Optimized dCBT with intermediate intensity conditioning, graft selection that prioritizes unit quality & CD34+ dose, CSA/ MMF prophylaxis with prompt therapy of PES & aGVHD, & letermovir CMV prophylaxis mitigates TRM in young & middle-aged adults. Thus, combined with relatively low relapse rates, the PFS & OS are high. Tocilizumab has been abandoned due to its notable association with delayed neutrophil engraftment & lack of survival advantage. Letermovir is highly effective completely preventing CMV infections & may contribute to the reduced aGVHD-related mortality recently (as compared to the pre-tocilizumab era), since avoidance of CMV antiviral therapy toxicities greatly facilitates aGVHD prevention & therapy. Given the rapid availability of cryopreserved unmanipulated CB grafts & frequent limitations of URD availability for minority populations, optimized dCBT is a highly attractive curative therapy for acute leukemias & other high-risk myeloid malignancies warranting prospective multi-center investigation.

Phase 1 Results of Bromodomain and Extraterminal Inhibitor PLX51107 in Combination with Azacitidine in Patients with Relapsed/Refractory Myeloid Malignancies.

Treatment outcomes in patients with relapsed/refractory (R/R) MDS and AML remains dismal. Based on both extensive pre-clinical data and emerging clinical data, treatment with bromodomain and extra-terminal domain inhibitors (BETi) is a potential approach for patients with high-risk myeloid malignancies. We conducted a phase I trial to study the safety and efficacy of PLX51107 (BETi) and azacitidine combination therapy in patients with R/R AML and high-risk (HR) MDS and studied mechanisms of resistance to the combination therapy. Thirty-seven patients with HR R/R MDS (n=4) and R/R AML (n=33) were treated. Sixteen patients (43%) had MECOM gene rearrangement and 7 other patients had TP53 mutation. Median prior number of therapies was three (range 1-9); 97% had received prior hypomethylating agent and 84% prior venetoclax. Overall response rate was 8/37 (22%): complete remission with incomplete platelet recovery (n=1); morphological leukemia-free state (n=2); hematologic improvement (n=5). The most common non-hematological toxicities were febrile neutropenia and pneumonia in 12 (32%) patients each; 6 patients (17%) had severe hyperbilirubinemia. RNA-Sequencing analysis of mononuclear cells harvested on treatment (day 3) vs pre-treatment showed significant changes in mRNA expressions in responders: downregulation of MYC, BCL2, IL7R and CDK6 and upregulation of HEXIM1, CD93, DCXR and CDKN1A. Immunoblot analyses confirmed reduction in protein levels of c-Myc, CDK6, BCL2 and BCL-xL, and induction of BRD4 and HEXIM1 protein levels in responders. In a heavily pre-treated patient cohort with R/R MDS and AML, PLX51107+ azacitidine was well-tolerated and resulted in modest clinical benefit.

Post-transplant cyclophosphamide and early mixed donor Chimerism in myeloid malignancies; a single-center experience

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option for high-risk myeloid malignancies. Post-transplant cyclophosphamide (PT-Cy) has proven to be effective for graft versus host disease (GVHD) prophylaxis. Given that graft-versus-tumor (GVT) effect plays a major role in reducing the risk of disease relapse, the application of PT-Cy must balance the risk of relapse. Mixed chimerism (MC) refers to a state of concurrent presence of recipient and donor cells post allo-HSCT which may precede relapse disease. ObjectiveWe investigated the impact of PT-Cy on early MC (EMC) and disease relapse in patients with a myeloid malignancy post allo-HSCT. Study designThis retrospective single-center study included patients that underwent allo-HSCT between 2015 and 2021. Patient and disease characteristics were collected from the electronic health records. EMC was defined as <95% donor cells at day 90–120 post allo-HSCT. ResultsA total of 144 patient that received an allo-HSCT were included in the study. One hundred and eight (75%) patients received PT-Cy as part of the GVHD prophylaxis regimen. The majority underwent allo-HSCT for acute myeloid leukemia (62%) or myelodysplastic syndrome (31%). Sixty-five percent received allo-HSCT from a matched unrelated donor transplant and 65% received a myeloablative conditioning regimen. A lower rate of chronic GVHD (p = 0.03) and a higher rate of EMC (p = 0.04) were observed in patients that received PT-Cy. PT-Cy was not associated with overall survival (OS) and relapse-free survival (RFS). Multivariable analysis identified measurable residual disease status (p = 0.003), hematopoietic cell transplantation-specific comorbidity index (p = 0.012) and chronic GVHD (p = 0.006) as independent prognostic variables for OS. AML-adverse risk (p = 0.004) and EMC (p = 0.018) were independently prognostic for RFS. While EMC overall was not significantly associated with higher risk of relapse, EMC was associated with shorter RFS within adverse-risk AML patients. ConclusionOur study shows that PT-Cy was associated with an increased risk of EMC. The predictive value of EMC for relapse remains unclear and may depend on the underlying disease, which should be validated in a larger cohort.

Busulfan, fludarabine, and melphalan are effective conditioning for pediatric and young adult patients with myeloid malignancies underdoing matched sibling or alternative donor transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for patients with high-risk myeloid malignancies. We present our 10-year experience (October 2012 to October 2021) of consecutive allo-HCT in patients with myeloid malignancies treated on the pediatric HCT service and conditioned with myeloablative targeted dose-busulfan (BU), fludarabine (FLU), and melphalan (MEL). Twenty-three children, adolescents, and young adult patients (CAYA) (median age 15.4years) with acute myeloid leukemia (AML, n = 17), myelodysplastic syndrome (MDS, n = 4), or chronic myeloid leukemia (CML, n = 2) underwent allo-HCT post-BU-FLU-MEL. Four patients had treatment-related AML/MDS. Donor/stem cell source was matched sibling donor (MSD) PBSC (n = 7), matched unrelated donor (MUD) PBSC (n = 2), umbilical cord blood (UCB) (n = 3), or haploidentical-BMT (n = 11). Risk stratification was low (n = 2), intermediate (n = 15), high (n = 3), and very high risk (n = 1). The two patients with CML had failed tyrosine kinase inhibitor therapies. With a median follow-up of 41.6months, the relapse rate is only 4.5% with an overall survival (OS) 100%, progression-free survival (PFS) 95.5%, and graft-versus-host-free-relapse-free survival (GRFS) 67.8%. The donor source and the acute graft-versus-host disease (GvHD) prophylaxis regimen significantly impacted grade II-IV aGvHD 66.7% versus 19.2% (p = .039) and chronic graft-versus-host-disease (cGvHD) 66.7% versus 0% (p = .002) in the patients receiving MSD or MUD PBSC compared to haplo-BMT, respectively, resulting in improved GRFS in haplo-BMT, 83.3% compared to 40% matched donor peripheral blood stem cell transplant (PBSCT) (p = .025). Our results demonstrate that BU-FLU-MEL is efficacious conditioning for disease control in young patients with myeloid malignancies undergoing MSD or alternative donor allo-HCT, but in the setting of PBSC grafts with cyclosporine A-methotrexate (CSA-MTX) GvHD prophylaxis, it results in an unacceptably high incidence of GvHD.

A Phase II Clinical Trial of "Off-the-Shelf" NK Cells with Allogeneic Stem Cell Transplantation to Decrease Disease Relapse in Patients with High-Risk Myeloid Malignancies

Background Acute myeloid leukemia, myelodysplastic syndrome, and TKI resistant CML continue to be among the most aggressive forms of blood cancer. Allogeneic hematopoietic stem cell transplantation (alloSCT) remains one of the most effective treatments available. Much of the benefit of AlloSCT is due to the immune-mediated graft-versus-leukemia effect to prevent relapse. Nevertheless, disease relapse remains the most important cause of treatment failure after alloSCT. A potential solution to reduce the risk of relapse may be found in Natural Killer (NK) cells, which have a demonstrable ability to eliminate leukemic and virally infected cells. However, naturally produced NK cells seen in patients early post-transplant are functionally impaired. A potential solution would be to provide donor-derived, expanded and activated NK cells in the peri-transplant setting. Natural killer cells are a unique class of lymphocytes, with cytotoxic, and immunoregulatory function which can mediate potent anti-leukemia effects. NK cells are regulated by Killer-cell Immunoglobulin-like Receptors (KIR) receptor-ligand interactions and mediate cytotoxicity. Alloreactive NK cells have been reported to enhance engraftment, reduce GVHD and prevent relapse of leukemia post transplant. This study will utilize NK cells from third party donors as adjuvant treatment to eradicate leukemia and prevent disease relapse after transplantation. Study Design and Methods Our clinical trial is registered as NCT05115630. Inclusion criteria includes age 18 to 65 years old, weighing at least 42 kg, with High risk AML, MDS, or TKI-resistant/intolerant CML who have either an HLA matched related donor, HLA matched unrelated donor, a haploidentical related donor, or a one antigen mismatched unrelated donor. HLA matching includes HLA A, B, C, and DR-B1. Exclusion criteria include patients who are HIV positive or has active hepatitis B or C, uncontrolled infections, liver cirrhosis, CNS involvement within 3 months prior to the transplant, or positive pregnancy test in a woman with child bearing potential. We will also exclude any patient with inability to comply with medical therapy or follow-up, patient with a known history of allergic reactions to any constituents of the product, including a known history of allergic reactions to cellular products or DMSO, other malignancy/cancer diagnosis <2 years ago. We will also exclude any patient requiring systemic corticosteroids with prednisone dose >10 mg or equivalent or who has KDS-1001 Donor specific antibodies (dsa) >5000 MFI units, or has KDS-1001 Donor anti-C1q positive. Statistical considerations and exploratory endpoints This is a single-arm Phase II trial of 3rd party natural killer (NK) cells at a fixed dose added to an allogeneic stem cell transplant for AML/MDS/CML. The goal is to reduce the risk of relapse of the malignancy post transplant, and to improve relapse free survival. The primary endpoint is the adverse event Failure = [graft failure, grade 3-4 GVHD, relapse or non-relapse death within 100 days post-transplant]. The stopping rule for time-to-failure (defined as graft failure, gr 3-4 GHVD, or non-relapse death) will be implemented to avoid waiting 100 days (3 months) to evaluate each patient to perform safety monitoring. The design will be based on T with 3 months follow-up, applying the design of Thall et al. Possibly right-censored values of T for patients treated in the trial will be monitored continuously throughout the trial. The accrual rate is expected to be approximately 2 patients per month, with each patient's follow-up continued until all patients have been followed for 3 months from their date of first NK cell administration. The distributions of time to failure and RFS time will be estimated using the method of Kaplan and Meier. Exploratory analyses with immunophenotyping for NK cell chimerism and persistence will be done as feasible. Immune reconstitution studies or additional future research may be performed.

Pilot Trial of IFN-γ and Donor Lymphocyte Infusion to Treat Relapsed AML and MDS after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Allogeneic stem cell transplantation (alloSCT) can cure high-risk myeloid malignancies. While non-relapse mortality has declined over the last several decades, more than half of alloSCT recipients die from recurrent leukemia, and little progress has been made in preventing or treating it. Only 10-20% of subjects with relapsed leukemia post-alloSCT become long-term survivors despite receiving donor lymphocyte infusions (DLI) or a second alloSCT. In mouse models, we identified that optimal CD4- and CD8- mediated graft-versus-leukemia (GVL) responses require interferon-gamma receptor (IFN-γR) expression on myeloblastic leukemias (Matte-Martone and Shlomchik, J Clin Invest. 2017). Other groups reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed post-alloSCT. These data support the hypothesis that IFN-γ sensitizes myeloid leukemia cells to alloreactive T cell-mediated killing. We conducted a phase I trial to evaluate whether a standard dose of IFN-γ can safely be administered to patients with relapsed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) post-alloSCT (NCT04628338). Methods: This investigator-initiated trial aimed to evaluate 1) the feasibility and safety of IFN-γ post alloSCT as monotherapy and combined with DLI; and 2) whether the standard dose IFN-γ is sufficient to upregulate IFN-γ-inducible molecules in myeloblasts in vivo. Major eligibility criteria included: 1) HLA-matched alloSCT recipient with relapsed AML/MDS; 2) measurable bone marrow (BM) myeloblasts (0.5-30%) by flow cytometry, 3) no increases in systemic immunosuppression for 4 weeks; and 4) no history of grade IV graft-versus-host disease (GVHD). The study had two stages: IFN-γ monotherapy (stage 1) with a fixed dose of IFN-γ (ACTIMMUNE®, Horizon Therapeutics) 100 mcg subcutaneously 3 times per week (outpatient self-injection) for 4 weeks and subsequent DLI combined with IFN-γ (stage 2) for 8 more weeks. Clinical responses were evaluated by BM pathology and donor chimerism at 4 weeks (pre-DLI), 12 weeks, and 6 months after the first dose of IFN-γ. BM, peripheral blood mononuclear cells, and plasma were collected before, 48-60 hours after IFN-γ, pre-DLI, and post-DLI. Phosphorylated STAT1 and HLA-ABC/DR, ICAM-1, and PDL-1 expression on BM leukemia blasts were measured by flow cytometry. Results: Four patients with post-transplant relapsed AML/MDS were enrolled (Table 1). All patients tolerated IFN-γ monotherapy well with anticipated injection site reactions, flu-like symptoms, and stable cytopenia. Three patients who received DLI achieved complete molecular and cytogenetics remissions and conversion to full donor chimerism coincident with steroid-sensitive GVHD. One of the three responders with donor T cell chimerism of >90% pre-treatment achieved a CR with IFN-γ monotherapy before DLI (Figure 1). All responders are currently alive and in complete remission without requiring additional anti-leukemia maintenance therapy (median follow-up 283 days; range 164-371). One patient who did not have available DLI and had poor donor T cell chimerism (8%) died from disease progression on day 139. BM blasts from pre-treatment phosphorylated pSTAT1 in response to in-vitro IFN-γ, demonstrating intact upstream IFN-γ R signaling. Blasts from all four patients increased HLA-DR expression 48-60 hours after IFN-γ therapy in vivo. We are performing single-cell RNAseq of BM cells to better understand IFN-γ -induced transcriptome changes across different maturation stages of leukemia blasts and donor-derived normal hematopoietic cells. Conclusion: IFN-g was safe in this pilot cohort of subjects with relapsed AML/MDS post-alloSCT. To our knowledge, this is the first time that IFN-γ has been tested on such subjects. In vivo, IFN-γ induced the predicted changes in myeloblasts. Most importantly, we observed durable CRs and conversion to 100% donor hematopoiesis with the IFN-γ/DLI combination, which is very promising for such poor-risk patients. These data provide a strong rationale for developing a phase II trial of IFN-γ/DLI for post-alloSCT relapse, and we are currently planning such a study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A021: Chimeric antigen receptor armored natural killer cell immunotherapy for osteosarcoma

Abstract Over the last decade, Chimeric Antigen Receptor based T cell (CAR-T) therapy has developed into an effective immunotherapy for several cancers, primarily limited to those of hematological origin. In other cancers, especially solid tumors, CAR-T cell therapies have several shortcomings and clinical success has been underwhelming. Challenges of CAR-T cell therapy include tumor immune evasion through loss of target antigen expression by tumor cells and inhibition of CAR-T cell function by tumor expressed inhibitory molecules. Natural killer (NK) cells present an alternative to T cells that could be more effective due to their ability to perform both antigen dependent and independent killing. NK cells can mediate the direct killing of transformed cells with reduced or absent MHC expression in addition to carrying out antibody dependent cell mediated cytotoxicity (ADCC) of cells bound by antibodies via the NK cell CD16A receptor. Engineering NK cells to express CARs will effectively enable them with additional antigen specific killing. Due to the multiple modalities for cancer cell killing, there is an increased interest in NK cells for cancer immunotherapy. As NK cells are not associated with graft versus host disease, neurotoxicity, long-term autoimmunity, nor cytokine release syndrome, they are more suited for use in allogeneic settings than T cells and have significant clinical potential for use as off-the-shelf products. However, previous publications and clinical trials have demonstrated that the use of unmanipulated NK cells to treat cancer is minimally effective, likely due to limited engraftment, little in vivo expansion or persistence, and suppression by the tumor microenvironment. NK cells activated and expanded with engineered feeder cells expressing membrane bound interleukin-21 (mbIL-21) and 4-1BBL have shown promising results clinically with high-risk myeloid malignancies and preclinically in several solid tumor models. Therefore, we hypothesize that activated/expanded CAR-NK cells that have been genetically edited can be used to successfully treat osteosarcoma, a disease for which patient outcome has not improved in over forty years. Our proposed objective is to evaluate the non-viral KI of several CARs, either alone or in combination, that optimally activate NK cell antigen-specific killing. Genetically engineered CAR-NK cells will be evaluated for enhanced therapeutic efficacy and safety in osteosarcoma models. Our preliminary data strongly supports the hypothesis that CAR-NK cell-based cancer immunotherapy can be fully realized using activated, genome engineered CAR-NK cells. Citation Format: Gabrielle Robbins, Kenta Yamomoto, Walker Lahr, Joseph Skeate. Chimeric antigen receptor armored natural killer cell immunotherapy for osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A021.

Impact on outcomes of mixed chimerism of bone marrow CD34+ sorted cells after matched or haploidentical allogeneic stem cell transplantation for myeloid malignancies.

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT), proposed to patients with high-risk myeloid malignancies, may ultimately fail because of disease relapse. Bone marrow (BM) CD34+ cells in Allo-HSCT recipients can be either re-emerging recipient malignant cells or donor cells attesting of hematopoietic reconstitution. In this context, investigating donor/recipient chimerism in the population of BM CD34+ sorted cells (BM-CD34+SC) was performed in 261 Allo-HSCT recipients (matched n = 145, haploidentical n = 65, matched unrelated n = 51) with myeloid malignancies. BM-CD34+SC chimerism was compared to that of whole peripheral blood (PB) cells as well as other Allo-HSCT-related parameters, and impact on relapse and survival was assessed. Thresholds of 98% donor cells for PB and 90% for BM-CD34+SC were found to allow relapse prediction. This was completed by the application of machine learning tools to explore the predictive value of these parameters in multidimensional models with repeated iterations. BM-CD34+SC mixed chimerism stood out with all these methods as the most robust predictor of relapse with a significant impact on disease-free and overall survivals even after haploidentical Allo-HSCT and/or PTCY administration. This marker therefore appears to be of great interest for the decision of preemptive treatment to avoid post-transplant relapse.

Abstract 2836: Genome engineered natural killer cell immunotherapy against osteosarcoma

Abstract Over the last decade Chimeric Antigen Receptor based T cell therapy (CAR-T) has developed into an effective immunotherapy for some cancers. Unfortunately, CAR-T cell therapies have several shortcomings and clinical success has primarily been limited to hematological cancers. Challenges of CAR-T cell therapy include tumor immune evasion through loss of target antigen expression by tumor cells and inhibition of CAR-T cell function by tumor expressed inhibitory molecules. Natural killer (NK) cells present an alternative to T cells that could be more effective due to their ability to perform both antigen dependent and independent killing. NK cells have demonstrated antigen specific killing when engineered to express CARs and NK cells also mediate the direct killing of transformed cells with reduced or absent MHC expression. Moreover, NK cells carry out antibody dependent cell mediated cytotoxicity (ACDD) of cells bound by antibodies via the NK cell CD16A receptor. Due to the multiple modalities for cancer cell killing, there is an increased interest in NK cells for cancer immunotherapy. As NK cells are not associated with graft versus host disease, neurotoxicity, long-term autoimmunity, nor cytokine release syndrome, they are more suited for use in allogeneic settings than T cells and have significant clinical potential for use as off-the-shelf products. However, previous publications and clinical trials have demonstrated that the use of unmanipulated NK cells to treat cancer is minimally effective, likely due to limited engraftment, little in vivo expansion or persistence, and suppression by the tumor microenvironment. NK cells activated and expanded with engineered feeder cells expressing membrane bound interleukin-21 (mbIL-21) and 4-1BBL have shown promising results clinically with high-risk myeloid malignancies and preclinically in several solid tumor models. Therefore, we hypothesize that activated/expanded CAR-NK cells that have been genetically edited can be used to successfully treat osteosarcoma, a disease for which patient outcome has not improved in over forty years. Our proposed objectives are to evaluate the baseline response of rested- and activated/expanded-NK cells against various osteosarcoma cell lines, knockout negative regulators of NK cell function (specifically, c-CBL, IL-1R8, and SMAD3), and implement several CARs alone or in combination that optimally activate NK cell antigen-specific killing. Genetically engineered CAR-NK cells will be evaluated for enhanced therapeutic efficacy and safety in osteosarcoma models. Our preliminary data strongly supports the hypothesis that CAR-NK cell-based cancer immunotherapy can be fully realized using activated, genome engineered CAR-NK cells. Citation Format: Gabrielle M. Robbins, Kenta Yamamoto, Joshua Krueger, Walker Lahr, Joseph Skeate, Branden Moriarity. Genome engineered natural killer cell immunotherapy against osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2836.

Long-term outcomes in patients with relapsed/refractory acute myeloid leukemia and other high-risk myeloid malignancies after undergoing sequential conditioning regimen based on IDA-FLAG and high-dose melphalan.

Allogeneic hematopoietic cell transplantation (alloHCT) remains the only curative option for relapsed/refractory acute myeloid leukemia and other high-risk myeloid malignancies. To improve alloHCT results in this setting, sequential regimens were designed as a strategy to lower tumor burden and quickly induce the graft-versus-leukemia effect. We analyzed long-term outcomes of a sequential regimen based on IDA-FLAG and high-dose melphalan, as set forth by the CETLAM cooperative group. This protocol yielded a high complete response rate (89%) and a lower cumulative relapse incidence (30% at five years) compared to other regimens. Five-year non-relapse mortality, however, reached 45%, with grade 3-4 acute graft-versus-host disease being the most frequent adverse event (a 100-day incidence of 29%). Altogether, 5-year overall survival was 25% in this group of patients with otherwise dismal prognosis. Long-term survivors enjoyed a good quality of life after a median follow-up of 68 months.

Single-agent 5-azacytidine as post-transplant maintenance in high-risk myeloid malignancies undergoing allogeneic hematopoietic cell transplantation.

Relapse is a major cause of treatment failure after allogeneic hematopoietic cell transplantation (allo-HCT) in myeloid malignancies. Additional strategies have been devised to further maximize the immunologic effect of allo-HCT, notably through maintenance therapy with hypomethylating agents such as 5-azacytidine (AZA). We conducted a single-center retrospective study to investigate the efficacy of AZA after allo-HCT for high-risk myeloid malignancies. All patients transplanted between Jan 2014 and Sept 2019 for high-risk acute myeloid leukemia (n = 123), myelodysplastic syndrome (n = 51), or chronic myelomonocytic leukemia (n = 11) were included. Patients who died, relapsed, or developed grade ≥ 2 acute graft-versus-host disease before day + 60 were excluded, as well as those who were eligible for anti-FMS-like tyrosine kinase 3 maintenance. Of the 185 included patients, 65 received AZA while 120 did not. Median age at transplant was 59years; 51.9% of patients were males. The median follow-up was 24months for both groups. Regarding main patient characteristics and transplantation modalities, the two groups were comparable. In multivariate analyses, there were no significant differences between the two groups in terms of 2-year cumulative incidence of relapse (HR = 1.19; 95% confidence interval (CI) 0.67-2.12; p = 0.55), overall survival (HR = 0.62; 95%CI 0.35-1.12; p = 0.12) and event-free survival (HR = 0.97; 95%CI 0.60-1.58; p = 0.91) rates. In conclusion,single-agent AZA does not appear to be an optimal drug for preventing post-transplant relapse in patients with high-risk myeloid malignancies. This study highlights the need for prospective studies of alternative therapies or combination approaches in the post-transplant setting.

A Genetic Disorder Reveals a Hematopoietic Stem Cell Regulatory Network Co-Opted in Leukemia

The molecular regulation of human hematopoietic stem cell (HSC) self-renewal and maintenance is of substantial interest, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. To better discern in vivo HSC function in humans, we have studied a rare genetic disorder due to MECOM haploinsufficiency that is characterized by neonatal aplastic anemia with an early-onset absence of HSCs in vivo.To establish a faithful model of MECOM haploinsufficiency, we performed CRISPR/Cas9 editing of MECOM in human hematopoietic stem and progenitor cells (HSPCs) and achieved predominantly heterozygous editing in phenotypic long-term (LT)-HSCs, as substantiated through both bulk and single cell assessments. Following MECOM editing, HSPCs showed a significant reduction in the number of phenotypic LT-HSCs as well as multipotential progenitor colonies in vitro, and had impaired engraftment following xenotransplantation into immunodeficient and Kit mutant mice. Next, we sought to use this model of MECOM haploinsufficiency to define the regulatory networks driven by MECOM that are critical for HSC maintenance. We therefore performed single-cell RNA sequencing on several thousand phenotypic LT-HSCs following MECOM editing and identified a list of 724 subtly but significantly differentially expressed genes compared to controls, including 322 genes that are downregulated after MECOM perturbation. Given the profound phenotypic effects associated with loss of MECOM and dysregulation of these gene sets, we sought to identify other cooperating factors that control the MECOM dependent gene network which underlies HSC self-renewal. To do so, we used integrative genomic approaches involving accessible chromatin and gene expression correlations, as well as long range chromatin interaction data across human hematopoiesis, to comprehensively define associations between genes and putative regulatory elements. Inspection of the nominated cis-regulatory elements controlling genes impacted by MECOM perturbation revealed significant enrichment for motifs and chromatin occupancy by several key cooperating transcription factors, including RUNX1, FLI1, and GATA2.In addition to the identification of cooperating transcription factors, we also discovered a strong binding motif enrichment for, and chromatin occupancy by, CTCF. CTCF is crucial to enable differentiation of LT-HSCs, and we found that MECOM regulated genes frequently had associated cis-regulatory elements that were occupied by CTCF. Moreover, these occupied cis-elements became more highly enriched for binding during hematopoietic differentiation. Chromatin conformation analysis revealed that the MECOM regulated genes with cis-elements bound by CTCF underwent chromatin reorganization and became more highly looped as LT-HSCs underwent differentiation, suggesting opposing functions of MECOM and CTCF in the regulation of the MECOM gene network. In light of these findings, we performed tandem perturbation of MECOM and CTCF and demonstrated rescue of LT-HSC loss with the dual perturbation, thereby illuminating a key role for MECOM in constraining CTCF-dependent chromatin reorganization that occurs as HSCs undergo differentiation.Finally, based on the observation that elevated MECOM expression is associated with high-risk myeloid malignancies, we investigated the role of the MECOM-regulated HSC gene network in acute myeloid leukemias (AML). Across three independent AML datasets, we found that the MECOM regulated gene network had an independent and strong prognostic prediction ability that enabled risk stratification beyond currently used approaches, including a variety of molecular criteria and the previously described LSC17 signature. To validate these correlative observations, we performed CRISPR/Cas9 editing of MECOM in the MUTZ-3 AML cell line that is characterized by MECOM overexpression. We found that MECOM editing results in a loss of CD34 + leukemia progenitors and that the same transcriptional network that we identified in LT-HSCs is similarly altered upon MECOM perturbation in these AML cells.Collectively, we use the study of a rare experiment of nature due to MECOM haploinsufficiency resulting in neonatal aplastic anemia to illuminate a gene regulatory network necessary for HSC self-renewal and maintenance that is co-opted in high-risk forms of AML. DisclosuresRegev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees. Sankaran: Forma: Consultancy; Ensoma: Consultancy; Novartis: Consultancy; Cellarity: Consultancy; Branch Biosciences: Consultancy.

A Phase 1 Dose Escalation Study of Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Patients with Myeloid Malignancies

A Phase 1 Dose Escalation Study of Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Patients with Myeloid Malignancies

FLAMSA-Busulfan-Melphalan as a Sequential Conditioning Regimen in HLA-Matched or Haploidentical Hematopoietic Stem Cell Transplantation for High-Risk Myeloid Diseases

Given the poor prognosis of relapsed/refractory myeloid malignancies, the concept of sequential conditioning before allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven to be an effective approach. We sought to evaluate a sequential scheme combining fludarabine, amsacrine, and cytarabine (FLAMSA) for cytoreduction, followed by reduced-intensity conditioning with busulfan and melphalan (FLAMSA-BuMel), which was designed to be suitable for both HLA-matched and haploidentical HSCT. This single-center retrospective study included 36 adult patients with high-risk myeloid malignancies who underwent allo-HSCT from HLA-matched (n=19) or haploidentical (n=17) donors. Along with the standard prophylaxis for graft-versus-host disease (GVHD), patients with a haploidentical donor received post-transplantation high-dose cyclophosphamide. A post-transplantation consolidation treatment with low-dose 5-azacytidine and prophylactic donor lymphocyte infusions was provided whenever possible. Thirty patients (83%) achieved complete remission on day +30. With a median follow-up of 30.0 months, the 2-year overall survival was 89% in the HLA-matched group versus 34% in the haploidentical group (P=.0018). The 2-year disease-free survival in these 2 groups was 68% and 34%, respectively (P=.013). At 2 years, the probability of relapse was 32% and 20%, respectively, and nonrelapse mortality was 0% and 58%, respectively (P=.0003). The leading cause of death was relapse in the HLA-matched group (3 of 19) and hemorrhagic events (5 of 17) in the haploidentical group, favored by significantly delayed platelet reconstitution and a severe GVHD context. These data confirm the feasibility of FLAMSA-BuMel as a sequential conditioning in allo-HSCT for high-risk myeloid malignancies. The use of bone marrow as the preferred graft source might reduce the incidence of acute GVHD and nonrelapse mortality in the haploidentical transplantation setting.