Combination Of Decitabine Research Articles

Abstract 5377: Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax

Abstract Introduction: Despite recent advances in the therapy of diffuse large B cell lymphoma (DLBCL), many patients are still not cured, and therefore new therapeutic combinations are needed. The anti-apoptotic B cell lymphoma 2 (BCL-2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32.3;q21.3), copy number alterations and gene amplifications; however, targeting BCL-2 with a selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a novel combination partner for venetoclax to improve response to therapy. Previously with dynamic BH3 profiling, we found that DNA hypomethylating agents (HMA) could increase BCL2 dependence in DLBCL cells. Here, we investigate the activity of the HMA decitabine with venetoclax in DLBCL. Methods: We utilized BH3 profiling, a functional assay to assess the mitochondrial priming of cells for apoptosis and the dependence of cells on various anti-apoptotic BCL-2 family proteins. Other standard techniques included Western blot, cell cycle (BrdU/7-AAD), CellTiter-Glo, and Seahorse XF cell mito stress test. The DLBCL cell lines (TMD8, HBL1, OCI-Ly3, OCI-Ly1, Karpas 422, SUDHL4, Toledo, OCI-Ly7) and double hit lymphoma (DHL) patient-derived xenograft cell lines were used to investigate the in vitro anti-cancer activity of decitabine and venetoclax. Results: Through BH3 profiling, we found heterogenous dependence of DLBCL, consistent with the variable expression of multiple anti-apoptotic proteins in DLBCL assessed by Western blot. DLBCL cells were less sensitive to single BH3 mimetic treatment (venetoclax, S63 (MCL-1i) and A133 (BCL-xLi)). Using dynamic BH3 profiling (which assesses the change in priming induced by ex vivo drug treatment), we found that decitabine primes DLBCL cells for apoptosis and increases BCL-2 dependence. Consequently, decitabine increases the sensitivity of DLBCL cells to venetoclax, as the combination of decitabine and venetoclax synergistically induces apoptosis and suppresses cell proliferation. Decitabine has previously been shown to increase TGF-β signaling in DLBCL by restoring the expression of a downstream target, SMAD1. Interestingly, we found that inhibition of TGF-β signaling only partially antagonizes the anti-cancer activity of decitabine, implicating other anti-cancer mechanisms of decitabine. Indeed, we found that decitabine also induces DNA damage and leads to cell cycle arrest in DLBCL cells. Furthermore, decitabine promotes activation of the key apoptotic effector proteins, BAK and BAX. Additionally, we found that this combination also suppresses oxidative phosphorylation, thus restricting the energy supply for DLBCL cells. Conclusions: The HMA decitabine sensitizes DLBCL cells to venetoclax in vitro through increasing apoptotic priming and BCL-2 dependence. This combination is worthy of further study in in vivo model systems. Citation Format: Matthew S. Davids, Fen Zhu, Stephen J. Chong, Jennifer Crombie, Liam Hackett, May C. Collins. Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax [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 5377.

Phase 2 study of ASTX727 (cedazuridine/decitabine) plus venetoclax (ven) in patients with relapsed/refractory acute myeloid leukemia (AML) or previously untreated, elderly patients (pts) unfit for chemotherapy.

7037 Background: ASTX727, is an oral formulation of the fixed dose combination of decitabine and cytidine deaminase inhibitor cedazuridine (100 mg/35 mg). We investigated whether a total oral therapy regimen of ASTX727+ven is feasible and safe. Methods: Pts aged ≥18 years (yrs) with relapsed/refractory AML (R/R) or pts with AML aged ≥ 75 or 18 -74 with comorbid conditions prohibiting intensive chemotherapy were eligible to participate (frontline-FL). Other eligibility criteria included adequate renal and hepatic function, and an ECOG performance status (PS) of≤2. ASTX727 is administered daily on days 1‐5 of each cycle and ven on days 1‐28 of the first cycle after a dose ramp up of 100-200-400 mg over 3 days (with tumor lysis prophylaxis precautions and with ven dose adjustments as needed). A bone marrow exam is performed on day 21±3 days and ven is held if blasts < 5% to allow count recovery. Cycles are repeated every 4-8 weeks and ven is administered for 21 days in subsequent cycles. Results: Between March 2021 and January 2022, 28 pts (15 FL and 13 R/R) have been treated on the study. The median age is 75 yrs (range, 47-90) with FL cohort 81 and R/R cohort 72. 9 FL pts (60%) were ≥80 and 5 (30%) 70-80 yrs. In R/R cohort 9 pts (69%) were 70-80 yrs. The median PS is 2 (range 0-3) and in the R/R cohort, the median number of prior treatments is 2 (range, 1-4). In the FL cohort 5 (33%) had normal and 6 (40%) a complex karyotype; 3 had other. In the R/R cohort, 15% had normal karyotype, 46% complex and 31% others. Mutations of note in the frontline cohort were RUNX1 (33%), ASXL1 (33%), DNMT3A (7%), TET2 (40%) and TP53 (20%). The overall response rate (ORR) including complete response (CR), CR with incomplete count recovery (CRi) and morphological leukemia free state (MLFS) in the FL cohort is 61% (4 CR, 4 CRi, 1 MLFS and 3 non-responders). 3 pts received only one day of therapy for severe adverse events unrelated to therapy (1 due to ischemic stroke, 1 septic shock and 1 debilitation) and were not evaluable for response. In the R/R cohort, the ORR was 45% (2 CR, 2 CRi, 2 MLFS with 5 non-responders and 2 not evaluable). The median number of cycles received is 2 (range, 1-5) for both cohorts. With a median follow-up of 5 months, the median survival for the FL cohort has not been reached (range, 0.6 – 7.3) and is 7.2 (range, 0.8-7.3) months for the R/R cohort. Grade 3 or higher adverse events directly attributable to therapy were mainly myelosuppression-related and included neutropenic infections in 3 (11%) and elevation of liver enzymes in 1 (4%) pt. Conclusions: Total oral therapy of ASTX727+ven is safe and feasible, particularly in the advanced elderly population, and demonstrates significant efficacy in pts unfit for chemotherapy both in the FL and R/R settings. Clinical trial information: NCT04746235.

A Study on the Effectiveness of Decitabine Combined with a Half-Dose Priming Regimen in the Treatment of Elderly Patients with Acute Myeloid Leukemia

Objective: To investigate the clinical effects of combining decitabine with a half-dose priming regimen in the treatment of elderly patients with acute myeloid leukemia. Methods: This study was conducted in Shaanxi Provincial People’s Hospital from January 2019 to January 2022. Sixty patients were recruited as the research subjects. The patients received different treatments and were randomly divided into two groups, with 30 cases in each group, one of which was treated with conventional priming regimen (control group), and the other was treated with decitabine combined with a half-dose priming regimen (study group). The two groups were compared and analyzed in terms of the effectiveness of treatment. Results: The rate of symptom relief in the study group was 96.67%, which was significantly higher than that in the control group (76.67%) (p < 0.05). Before treatment, there was no significant difference in the quality-of-life scores between the two groups, with p > 0.05. The patients in the study group had significantly longer disease-free survival and overall survival than those in the control group, with p < 0.05. The effectiveness of treatment in the study group was also better. Conclusion: The use of decitabine in combination with a half-dose priming regimen for the treatment of elderly patients with acute myeloid leukemia is effective in improving patients’ quality of life, relieving symptoms, and prolonging their survival.

CLO22-035: Treatment Outcome of Ruxolitinib and Decitabine Combination Chemotherapy Among Transplant Ineligible Advanced Stage Myeloproliferative Neoplasm: A Meta-analysis

"CLO22-035: Treatment Outcome of Ruxolitinib and Decitabine Combination Chemotherapy Among Transplant Ineligible Advanced Stage Myeloproliferative Neoplasm: A Meta-analysis" published on 31 Mar 2022 by National Comprehensive Cancer Network.

Chidamide and Decitabine in Combination with a HAG Priming Regimen for Acute Myeloid Leukemia with TP53 Mutation.

We analyzed the treatment effects of chidamide and decitabine in combination with a HAG (homoharringtonine, cytarabine, G-CSF) priming regimen (CDHAG) in acute myeloid leukemia (AML) patients with TP53 mutation. Seven TP53 mutated AML patients were treated with CDHAG. The treatment effects were assessed using hemogram detection and bone marrow aspirate. The possible side effects were evaluated based on both hematological and non-hematological toxicity. Four of the seven patients were classified as having achieved complete remission after CDHAG treatment; one patient was considered to have achieved partial remission, and the remaining two patients were considered in non-remission. The overall response rate (ORR) to CDHAG was 71.4%. Regarding the side effects, the hematological toxicity level of the seven patients ranged from level III to level IV, and infections that occurred at lung, blood, and skin were recorded. Nausea, vomiting, liver injury, and kidney injury were also detected. However, all side effects were attenuated by proper management. The CDHAG regimen clearly improved the ORR (71.4%) of TP53-mutated AML patients, with no severe side effects.

Patients with AML-MRC benefit from decitabine in combination with low-dose G-CSF, cytarabine and aclarubicin: A single center cohort study

Patients with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) are insensitive to conventional chemotherapy and, therefore, have a poor prognosis. To evaluate the clinical efficacy and safety of low-dose decitabine in combination with small-dose CAG regimen (D-CAG regimen) in treating AML-MRC, a total of 80 patients with newly diagnosed AML-MRC from September 2015 to January 2020 in our center were included in the study. Amongst these patients, 43 and 37 patients received two courses of the D-CAG and CAG regimens, respectively. The complete remission (CR) and complete remission with incomplete blood count recovery (CRi) rate in the D-CAG group was higher than that of the CAG group (62.2% vs. 48.8%, P = 0.013). Among patients with less than 9 months of myelodysplastic syndrome (MDS) history and poor karyotypes, the (CR+CRi) rate of the D-CAG group was statistically higher than that of the CAG group. Except for patients receiving hematopoietic cell transplantation, among patients with less than 9 months of MDS history, the D-CAG group showed a better probability of overall survival than the CAG group did. In conclusion, patients with AML-MRC may benefit from the D-CAG regimen as an induction therapy, especially patients with less than 9 months of MDS history or with poor karyotypes.

Inhibitory Effect of Ascorbic Acid on Myelodysplastic Syndrome Cells and Its Mechanism

To investigate the inhibitory effect of ascorbic acid single or combination of decitabine on tumor cells of myelodysplastic syndrome (MDS) and explore its related mechanism. The human MDS cell lines SKM-1 and MUTZ-1 were treated with different concentrations of ascorbic acid, and the cell proliferation activity was detected by the CCK-8 assay. The reactive oxygen species (ROS) level, labile iron pool (LIP), cell cycle, and apoptosis of SKM-1 and MUTZ-1 cells were detected by flow cytometry. The control group, ascorbic acid monotherapy group, decitabine monotherapy group, and combination group of ascorbic acid and decitabine were set up, the cell proliferation activity and apoptosis were detected in each group. High-dose ascorbic acid could reduce the cell proliferation activity of SKM-1 (R=0.886, p=0.000) and MUTZ-1 (R=0.880, p=0.000). With the increase of ascorbic acid concentration, the ROS level in SKM-1 and MUTZ-1 cells increased (r=0.816, r=0.942), the proportion of cells stagnation in G2 phase increased (r=0.970, p=0.000; r=0.962, p=0.000), the proportion of surviving cells decreased (r=-0.966, p=0.000; r=-0.952, p=0.000), and the apoptosis cells significantly increased (r=0.966, p=0.000; r=0.958, p=0.000). Nevertheless, the level of LIP showed no significant changes. After the combined application of ascorbic acid and decitabine, MDS tumor cells showed decreased proliferative activity and increased apoptosis compared with single-agent ascorbic acid and decitabine group. High-dose ascorbic acid shows a cytotoxic effect on MDS tumor cells, inhibiting cell proliferation and increasing apoptosis. Ascorbic acid combined decitabine have a synergistic effect of anti-MDS tumor cells.

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells.

BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

The Application of Decitabine in Combination with G-CSF, Low-Dose Cytarabine and Aclarubicin in AML-MRC: A Single Center Case Control Study

▪Background: It is difficult for AML-MRC patients to achieve complete remission (CR) and these patients develop recurrence and die of either disease progression or associated complications. The CAG regimen (cytarabine, aclarubicin and G-CSF) has been widely used in treating patients with AML-MRC in Asia. Decitabine (DAC) was approved to the treatment of MDS and reported to achieve higher response rate (67%) in AML with unfavorable-risk cytogenetics. Several studies reported low dose DAC in combination with chemotherapy to treat AML.Purpose:To evaluate the clinical efficacy and safety of low-dose decitabine in combination with small-dose CAG regimen (D-CAG regimen) in the treatment of AML-MRC, compared to CAG regimen.Methods:A total of 80 patients with newly diagnosed AML-MRC from September 2015 to January 2020 in our center were included in the study. 43 cases were initially treated with G-CSF for priming, in combination with cytarabine of 10-mg/ m 2q12h for 14 days and aclarubicin of 20 mg/day for 4 days (CAG regimen) and other 37 cases were initially treated with decitabine of 20 mg/m 2 for 5 days and small-dose CAG regimen (cytarabine of 10-mg/ m 2q12h for 7 days, aclarubicin of 10 mg/day for 4 days, and G-CSF for priming (D-CAG regimen). After induction chemotherapy, the patients who achieved CR received consolidation chemotherapy or hematopoietic stem cell transplantation (HSCT).Results:Among a total of 80 patients, the median age was 55 years (18-69 years) and 32 of them were male. Baseline characteristics of patients between D-CAG group and CAG group showed no significant differences (table 1). For the overall AML-MRC patients, the MLFS rate of D-CAG group was higher than the MLFS rate of CAG group after two courses of D-CAG regimen (62.2% v. s. 48.8%, χ2 =8.727, P=0.013, bilateral). Seven patients in CAG group did not response and then received D-CAG regimen as induction therapy, and five of them achieved MLFS (5/7) and one achieved PR (1/7). Among the population with less than 9 months of AML-MRC and/or MDS history, the MLFS rate (74.1%, 20/27) of D-CAG group was statistically higher than the MLFS rate (42.9% 15/35) of CAG group (74.1% v. s. 42.9%, χ 2 =1.909, p=0.008). Compared to the CAG group, the high-risk patients classified according to SWOG criteria in D-CAG group achieved a better MLFS rate (80.0% v. s. 26.1%, χ 2 =11.392, P=0.003, bilateral). Except patients receiving HSCT, the probability of OS and LFS for patients between D-CAG group and CAG group did not show any significant difference, but among the population with less than 9 months of AML-MRC and/or MDS history, the DCAG group showed a better probability of OS than the CAG group (58.3%±18.6% v. s. 11.3%±10.3%, p=0.006).Conclusion: In conclusion, patients with AML-MRC have a poor prognosis, and might benefit from D-CAG regimen as the induction therapy. For patients with less than 9 months of AML-MRC/MDS history or with poor karyotypes, the MLFS rate for patients in D-CAG group was higher than patients in CAG group. DisclosuresNo relevant conflicts of interest to declare.

Decitabine in Combination with Fludarabine and Cyclophosphamide As Lymphodepletion Regimen Followed By CD19/CD22 Bispecific Targeted CAR-T Therapy Significantly Improves Survival in Relapsed/Refractory B-ALL Patients: A Pilot Study

Background: CD19/CD22 bispecific targeted chimeric antigen receptor T cell (CAR-T) therapy has achieved impressive progress in patients with relapsed/refractory B cell acute lymphoblastic leukemia (R/R B-ALL), with high rates of complete remission (CR). However, T cell exhaustion caused by de novo DNA methylation restricts the capacity of CAR-T. Decitabine (DAC), a DNA methyltransferase inhibitor, has been demonstrated to reverse exhaustion-associated DNA-methylation programs, promote the rejuvenation of CAR-T cells and enhance anti-leukemic effect of CAR-T cells in vitro. To date, there are limited reports about the use of DAC as a part of lymphodepletion therapy before CAR-T cell therapy. Here, we report efficacy and safety of DAC in combination with fludarabine and cyclophosphamide (FC) regimen followed by CD19/CD22 CAR-T cell therapy for patients with R/R B-ALL.Method:We conducted a phase 1/2 clinical trial to investigate the efficacy and safety of CD19/CD22 CAR-T in the treatment of R/R B-ALL (NCT03614858). Fourteen patients were treated with DAC (total dose 100mg/m 2 in 3 days) followed by FC regimen (fludarabine 30mg/m 2 × 3d and cyclophosphamide 300mg/m 2 × 3d) (DAC group), while twelve patients received FC regimen prior to CAR-T cell infusion (CON group). A total of 1 or 2 × 10 7 CAR-T cells/kg were infused at dose escalation.Results: Baseline characteristics of patients in both groups had no significant differences except previous hematopoietic stem cell transplantation (HSCT). There were more patients with relapse after HSCT in DAC group (42.9% versus 0%, P=0.017). All patients did not achieve remission before lymphodepletion. The day 28 CR rates were 100% in DAC group and 91.7% in CON group. Furthermore, minimal residual disease (MRD) negative CR rates were 71.4% and 58.3%, respectively (P = 0.683). There was no significant difference in the proportion of nontransplant patients after CAR-T treatment between two groups. Among the nontransplant patients after CAR-T infusion in DAC group, 16.7% (1/6) of patients relapsed at 4 months. However, among 4 nontransplant patients in CON group, 1 patient achieved NR after CAR-T therapy and 3 patients relapsed at 1.5, 5, and 10 months. There were significant differences in overall survival (OS) and leukemia-free survival (LFS) between two groups: 3-year OS, 88.9% (DAC) versus 33.3% (CON), P = 0.01 and 3-year LFS, 92.3% (DAC) versus 21.8% (CON), P = 0.002. Multivariable analysis showed that addition of DAC to the lymphodepletion regimen was associated with better OS (hazard ratio [HR] 0.107, [95% CI, 0.013-0.875], P = 0.037) and LFS (HR 0.081, [95% CI, 0.01-0.65], P = 0.018). Cytokine release syndrome was observed in all patients.Conclusion: In summary, DAC in combination with FC regimen followed by CD19CD22 CAR-T cells was feasible and well tolerated. Our study demonstrated DAC combined with FC was an independent prognostic factor correlated with better survival in relapsed/refractory B-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Clinical Efficacy Analysis of the Low-Dose Decitabine in Combination with Cytarabine, All-Trans Retinoic Acid and Granulocyte Colony-Stimulating Factor for Patients with MDS-EB

▪AbstractPurpose:Treatment of patients with myelodysplastic syndromes with excess blasts (MDS-EB) remains a great challenge. In this study, we evaluated the tolerance and efficacy of the combination of low-dose decitabine, cytarabine, all-trans retinoic acid and granulocyte colony-stimulating factor (DLAAG) for MDS-EB patients.Methods:A total of 18 patients with MDS-EB were enrolled in this study and 1 patient who did not follow-up the bone marrow evaluation was excluded from analysis. This study is registered at ClinicalTrials.gov, NCT03356080.The DLAAG regimen consisted of subcutaneous injection of decitabine (0.1-0.2mg/kg, d1-3/w, lasted for 1-3 weeks), cytarabine (6-15mg/m 2,q12h,d1-10), oral all-trans retinoic acid (45mg/m 2/d, d4-6; 15mg/m 2/d, d7-20) and G-CSF (300ug, from d0 until neutrophil count recovery to 20×10 9 cells/L).Results:Patient characteristicsA total of 18 patients (11 males, 7 females) with a median age of 61 years (range 45 to 88 years) were enrolled in our study. The clinical characteristics of the patients were shown in Table 1.Response and survivalThe clinical outcome and overall survival (OS) of patients were summarized in Table2 including 1 could not evaluable after the first protocol. As shown in Table3, after one course of DLAAG, the CR and OR rate was 55.56% and 77.78% respectively. In addition, 3 patients suffered the disease progression (PD) (n=3,16.67%). We found that those who achieved the bone marrow remission after DLAAG had significantly pro-longed OS than others (p=0.0001, Fig.1). The Kaplan-Meier analysis revealed the median overall survival for all patients enrolled was 15 months (Fig.2A), and the relapse-free survival (RFS) for patients achieving CR or CRm was 11 months (Fig.2B).Genetic mutations and response to DLAAGPaired samples (pre- and post-treatment) of the objects were tested for gene mutations which were recurrently mutated in myeloid malignancies, except 1 patient who was failure to perform bone marrow review. 2 patients (11.1%) had no genetic or cytogenetic abnormalities either at baseline or during therapy (they were tested at baseline and d28). Most patients (15 out of 18) had at least one somatic mutation. In this latter group, only 1 patient acquired specific mutation of IDH2 during the therapy (it was tested on d28, data was not shown), while all the other genes were mutated in patients at baseline. Collectively, the most frequently mutated genes at diagnosis were RUNX1 (n=5, 29.4%), ASXL1 (n=4, 23.5%), as well as the overexpression of WT1 (n=4, 23.5%), and the others with an incidence of more than 10% were TET2(n=3,17.6%),TP53 (n=2, 11.8%), EZH2 (n=2, 11.8%), STAG2 (n=2, 11.8%), SF3B1 (n=2, 11.8%) and U2AF1 (n=2, 11.8%).Genes less frequently mutated such as CEBPA,CUX1 et al. were not shown. Remarkably, the proportion of patients with the above gene mutations showing no significant increasing during therapy as compared to baseline, the clones of WT1/TET2/TP53/EZH2/STAG2 can decrease or even disappear(Fig.3A).Moreover, we noted the complete response in patients with ASXL1, TP53 mutations or WT1 overexpression despite the concomitant presence of complex cytogenetic at diagnosis, whereas patients with the EZH2 mutation were less likely to respond (Fig.3B).Subgroup analysisA subgroup analysis based on the 2016 WHO classification revealed an association of EB-I patients with longer OS(P=0.002) (Fig. 4A). On the other hand, based on the IPSS score our study revealed that the lower-risk group patients have better OS (p= 0.0054) (Fig. 4B).In addition to this, we also compared the OS in different groups of age and karyotype, but there were no statistical difference (Fig. 4C-D).Safety and TolerabilityAll patients who received the therapy were eligible for toxicity evaluation. Given the patient-population enrolled and treatment regimen, myelosuppression occurred in all patients (Table 4). The grade 3 to 4 hematological toxicities including neutropenia and thrombocytopenia were common after treatment. Febrile neutropenia occurred in 77.8% of the patients.Conclusion: This study suggested the novel combination of LD-DAC, Ara-C, ATRA and G-CSF might be an optimal introduction therapy for patients with MDS-EB, and this combination warrants further investigation in larger trials. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combination of Decitabine and ATRA in Newly Diagnosed Myelodysplastic Syndromes Subtype EB-Interim Analysis of a Multicenter, Randomized, Open-Label Trial

Background: HMAs are mainstay treatment of higher-risk myelodysplastic syndromes (MDS). However, clinical outcomes of patients treated with decitabine (DEC) monotherapy were far from satisfactory with an overall response rate (ORR) of 33%-55.4% and an overall survival (OS) of 17.7-22 months. Some clinical researches reported that the addition of all-trans retinoic acid (ATRA) to DEC increased response rate and prolonged survival of MDS and elderly acute myeloid leukemia (AML) patients. Our data showed ATRA enhanced the cytotoxic effect of DEC on MDS via activating RARα-Nrf2 complex (2021 EHA abstract EP891). These findings suggested that addition of ATRA to DEC in treatment-naive patients may improve response rate based on the synergetic function. We therefore conducted a study of combination of DEC and ATRA in MDS subtype excessive blasts (EB) patients.Methods: In this randomized, multicenter, open-label trial, patients with newly diagnosed MDS subtype EB based on the 2016 WHO classification from 7 different tertiary medical centers in China were included. Patients were randomized 1:1 to receive either oral ATRA (25mg/m 2/day on days 1-28) plus DEC (20 mg/m 2 daily on days 1-5) or DEC monotherapy（Figure 1）. The primary endpoint was overall response rate ORR, defined as complete remission (CR), partial remission (PR), marrow complete remission (mCR), or hematological improvement (HI). Key secondary endpoints were mCR, HI, overall survival (OS), and progress free survival (PFS). Response was assessed after completion of four cycles of treatment. For patients who bridged to allo-HSCT later on and did not complete four cycles of treatments, response was defined as best response ever observed before receiving allo-HSCT. Here, we report results of the interim analysis.Results: Between May 2018 and July 2021, 165 patients were randomly allocated into either DEC plus ATRA (n=82) or DEC monotherapy (n=83). 63.6% of patients were male and 36.4% were female, with a median age of 62 years (range, 19 to 81 years). 38.8% of patients had EB1 and 61.2% had EB2. As of July 31, 2021, 126 patients were available for the assessment of treatment results.After a median follow up of 9.6 months, median number of courses on treatment was 4 courses (range, 1-14), 61 in DEC plus ATRA arm and 65 in DEC arm . OR was achieved in 85.2% of DEC plus ATRA patients compared to 56.9% in DEC monotherapy (p<0.001). mCR rate was 73.8% in patients treated with DEC plus ATRA and 53.8% in those with DEC monotherapy (p=0.02). HI rate was 63.9% and 44.6% in patients with DEC plus ATRA and DEC monotherapy, respectively (p=0.03). The median OS and PFS were 18.8 months and 13.5 months for DEC plus ATRA arms, 19.2 months and 13.0 months for in DEC arm, respectively.72.7% patients developed at least one adverse event (AE) during the trial, 73.3% in the DEC plus ATRA arm and 72.0% in DEC arm, respectively. Grade 3/4 Hematological toxicity occurred in 73.68 % of DEC plus ATRA arm and 76.92 % of DEC arm. Hyperlipidemia occurred in 31 patients in DEC plus ATRA arm (3.2% grade 3/4), and 18 (no grade 3/4) in DEC arm (p<0.001). Incidence of headache was higher in DEC plus ATRA arm (14.7% vs 4.0% in DEC arm, respectively, p<0.001). No other statistically significant differences were observed in the incidence of treatment-related AEs between the two groups. No early death occurred.Conclusion: Combination of DEC and ATRA achieved an OR rate of 85.2% in MDS subtype EB. Enrollment is ongoing to assess its efficacy and safety in treating EB. This therapy may be a new treatment option for EB subjects. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy and Safety of Decitabine Combined with IA Regimen in the Treatment of Newly Diagnosed Acute Myeloid Leukemia

▪Objective: To evaluate the efficacy and safety of decitabine combined with IA regimen in the treatment of newly diagnosed acute myeloid leukemia.Methods: From September 1, 2013 to October 18, 2019, 164 newly diagnosed acute myeloid leukemia patients who received IA or decitabine combined with IA induction chemotherapy who were hospitalized in the Department of Hematology of the First Affiliated Hospital of Sun Yat-sen University were enrolled. The efficacy and side effects of treatment were analyzed.Results: The complete remission rate of decitabine combined with IA regimen chemotherapy group (n=88) and IA regimen chemotherapy group (n=76) was 83.0% vs. 68.4% (P=0.029, Fig 1). Subgroup analysis (table 1) showed that age ≥40 years old, WBC＜10*10^9/L, Hb＞85g/L, PLT≥50*10^9/L, MCV≥98fL, ratio of bone marrow immature cells ≤45%, NCCN intermediate-risk or high-risk group, patients with FLT3ITD mutation had a higher CR rate in the decitabine combined with IA regimen group. Multivariate analysis showed that combined decitabine was an independent favorable factor affecting the CR rate (OR 3.559, 95% CI: 1.554-8.151, P=0.003). Compared with the IA group, patients in the decitabine combined with IA group took longer to rebuild the granule system (20 days vs 19 days, P=0.026), and the incidence of infection was higher (93.2% vs 78.8%, P=0.028) (table 2).Conclusion: Compared with the IA regimen, the decitabine combined with the IA regimen significantly improves the induction chemotherapy response rate of newly diagnosed non-M3 AML patients, especially for patients with the following characteristics: age ≥ 40 years old, WBC <10*10^ 9/L, Hb＞85g/L, PLT≥50*10^9/L, MCV≥98fL, bone marrow immature cell proportion ≤45%, NCCN risk stratification medium-risk or high-risk group, FLT3ITD mutation. After combining with decitabine, the patient's granular bone marrow suppression increased. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Phase 1 Study Evaluating ASTX727 (decitabine and cedazuridine) and Venetoclax Combination Therapy in Newly Diagnosed AML Patients Unfit for Intensive Induction Chemotherapy

A Phase 1 Study Evaluating ASTX727 (decitabine and cedazuridine) and Venetoclax Combination Therapy in Newly Diagnosed AML Patients Unfit for Intensive Induction Chemotherapy

Efficacy of Oral Decitabine/Cedazuridine (ASTX727) in the CMML Subgroup from the Ascertain Phase 3 Study

Abstract text:Background/Introduction:Chronic Myelomonocytic Leukemia (CMML) is an uncommon MDS/MPN overlap syndrome that has historically been included under the umbrella of myelodysplastic syndromes (MDS) for clinical trial and treatment. As a result, DNA methyltransferase inhibitors (DNMTi) such as decitabine or azacitidine have been the established standard of care for the treatment of CMML. The oral bioavailability of these agents has been limited due to rapid degradation by cytidine deaminase (CDA) in the gut and liver so treatment has required intravenous infusion or subcutaneous injections daily for 5-7 days every month (m) adding significant burden to older cancer patients due to daily time commitment and travel to treatment centers. In the context of pandemic SARS-CoV-2, parenteral therapy also increases contact with medical settings with increased infection risk. Oral decitabine 35 mg/cedazuridine 100 mg (ASTX727) is an oral fixed dose combination of decitabine and the CDA inhibitor cedazuridine that produced equivalent exposure (99%; 90% CI 93% to 106%) to IV decitabine 20 mg/m 2 in a randomized cross-over study (Garcia-Manero et al, ASH 2019), and Median overall survival (mOS) for the entire study population in the ASCERTAIN study was approximately 32 months (Savona, 2021). Here, we present outcome data for this study for the enrolled subpopulation of patients with CMML.Methods:We used a randomized cross over design in which patients were randomized in the first 2 cycles 1:1 to either Sequence A: (decitabine 35 mg/ cedazuridine 100 mg in Cycle 1 followed by IV decitabine at 20 mg/m 2 in Cycle 2), or Sequence B: (IV decitabine in Cycle 1 followed by oral decitabine/cedazuridine in Cycle 2). We conducted an intra-patient comparison of decitabine PK (primary PK endpoint: decitabine AUC equivalence over 5 days of dosing). Cycles were repeated every 28 days (unless delays were needed). All patients received oral decitabine/cedazuridine in Cycles 3 and above until disease progression or unacceptable toxicity. Patients were eligible per the FDA-approved label of IV decitabine (MDS patients by FAB classification including CMML, or MDS IPSS Intermediate-1, 2 or high-risk patients). Clinical endpoints were best response according to International Working Group (IWG) 2006 response criteria, transfusion independence for at least 8 or 16 consecutive weeks, overall survival, and safety. Adverse events (AEs) were graded by Common Terminology Criteria for Adverse Events (CTCAE) v 4.03.Results:Of the 133 patients enrolled and treated in ASCERTAIN, 16 (12%) had a diagnosis of CMML with demographics and as follows: median age 71.5 years, 69% Male/31% Female, median weight 87kg (range 65-124), 25% ECOG 0, 75% ECOG 1. Population disease characteristics were: 19% poor or intermediate risk cytogenetics, with median baseline hemoglobin 90 g/L, neutrophils 1.27 X 10 9/L, platelets 84 x 10 9/L, bone marrow blasts 5%, with 38% RBC transfusion dependent. Patients received a median of 7 cycles of therapy (range 3-24). Treatment-emergent adverse events of CTCAE Grade 3 or higher in > 10% of patients, independent of relationship to ASTX727, were cytopenias (neutropenia [69%], thrombocytopenia [63%], anemia [56%], leukopenia [19%]), febrile neutropenia (31%), fatigue (13%). Two patients (12.5%) had Complete Responses (CR), 8 (50%) had marrow CR ([mCR], including 3 (19%) with hematologic improvement (HI); Overall Response rate (ORR) [CR + PR+ mCR + HI] was 75%. Of six patients with baseline RBC transfusion dependence 3 (50%) became transfusion independent. Leukemia-free survival was 28.2 months and after a median follow up of more than 33 months, median overall survival had not been reached. Two patients (13%) went on to Hematopoietic Stem Cell Transplant (HCT).Conclusions:In the overall study, oral decitabine/cedazuridine delivered equivalent PK exposure to 5 days of IV decitabine 20mg/m 2 with a resultant clinical activity safety and efficacy profile in CMML patients consistent with the published literature (e.g Zeidan, et al 2017) and the Phase 2 experience. The use of oral decitabine/cedazuridine is a reasonable approach in CMML patients.

Mitochondrial DNA Mutations Distinguish Individual Donor- and Recipient-Derived Immune Cells Following Matched Unrelated Allogeneic Stem Cell Transplantation

▪Reconstitution of donor hematopoiesis after allogeneic hematopoietic stem cell transplantation (HSCT) forms the basis for effective graft-versus-leukemia responses, but mixed chimerism is not an infrequent outcome. How the donor and host hematopoietic system interact under conditions of mixed chimerism remains incompletely understood. Multi-modal single cell sequencing platforms are increasingly available and provide information regarding cell identities and interactions at high resolution. However, the analysis of post-transplant immune reconstitution requires consistent distinguishing of donor- and recipient-derived cells, which for sparse single cell sequencing data until now has remained a challenge.Recently, mitochondrial DNA (mtDNA) mutations have been recognized for their potential as personal genetic barcodes that can be detected with mitochondrial single cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq). We hypothesized that individual-specific mtDNA mutations could provide a sensitive and robust approach for distinguishing donor- from recipient-derived cells, and therefore tested this approach on bone marrow (BM) samples from patients with relapsed acute myeloid leukemia (AML) post-HSCT. We employed ATAC with select cell surface antigen profiling by sequencing (ASAP-seq), which enables the detection of mtDNA mutations within distinct surface marker-defined cell populations alongside chromatin accessibility. We selected serial samples collected from the ETCTN 10026 study, which tested combined decitabine (days 1-5, every 4 weeks, start cycle 0) and ipilimumab (day 1, every 4 weeks, start cycle 1) in relapsed AML post-HSCT. We focused on 13 samples (study entry, on treatment and disease progression) from 3 patients: AML1012 (HSCT from a matched related donor [MRD]), and AML1010 and AML1026 (matched unrelated donor [MUD]-HSCT).In total, we obtained 33,943 ASAP-seq profiles, including 3,283 single T cells. While clustering using single cell chromatin profiles alone only allowed identification of either CD4 + or CD8 + T cells, integration with surface marker expression enabled more detailed annotations of 8 T cell subpopulations and NK cells. Further, phenotypically distinct subpopulations such as CD57 + CD4 + and CD8 + T cells shared highly similar chromatin profiles, and 11.1% of CD4 + and 33.7% of CD8 + T cells would have been mislabeled based on clustering of chromatin profiles alone. Thus, ASAP-seq identified T cell subsets with markedly improved accuracy and resolution than scATAC-seq alone.Upon evaluation of mtDNA mutations to discriminate donor- and recipient-derived single T cells, we found that this was unreliable for MRD-HSCT (AML1012), but highly robust in the setting of MUD-HSCT (AML1010, AML1026), consistent with maternal inheritance of mitochondrial genomes. For the latter two patients, we identified 48 donor- and 26 recipient-specific mtDNA mutations, all with high heteroplasmy (range 82 - 99%). Presence of donor- and recipient-derived mtDNA mutations was mutually exclusive, and recipient-specific mtDNA mutations were also detectable in AML cells. Clinical bulk and mtDNA mutation-based single T cell chimerisms were highly correlated (r = 0.97).AML1010 had sustained complete T cell chimerism (>97%) during study treatment. In AML1026, the mtDNA mutation-based T cell chimerism rose from 55% to 71% after 1 cycle of decitabine and then remained stable until disease progression 3 months later. This was associated with increased percentage of donor-derived CD4 + T cells (45% [study entry] vs. 71% [after 1 cycle of decitabine], p < 0.01), while donor-derived CD8 + T cells remained unchanged at 76%. Across all studied timepoints in AML1026, donor versus recipient skewing was also highest in CD4 + T cell subsets, with fewer naïve (20% vs. 31%, p < 0.01) but more donor-derived CD57 + CD4 + T cells (13% vs. 3%, p < 0.01).We demonstrate that mtDNA mutations can discriminate between donor- and recipient-derived single cells, enabling detection and in-depth characterization of chimeric immune cell dynamics after MUD HSCT. This approach will allow to systematically dissect conditions of mixed chimerism in the post-transplant setting with larger studies. DisclosuresDeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Incyte: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Sankaran: Cellarity: Consultancy; Forma: Consultancy; Novartis: Consultancy; Branch Biosciences: Consultancy; Ensoma: Consultancy. Soiffer: Jasper: Consultancy; Jazz Pharmaceuticals, USA: Consultancy; Precision Biosciences, USA: Consultancy; Juno Therapeutics, USA: Other: Data Safety Monitoring Board; Kiadis, Netherlands: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics, USA: Consultancy; Gilead, USA: Other: Career Development Award Committee; NMPD - Be the Match, USA: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy. Garcia: Genentech: Research Funding; Prelude: Research Funding; Pfizer: Research Funding; AstraZeneca: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company. OffLabel Disclosure:ipilimumab to modulate anti-leukemia immunity in the post-transplant and transplant-naive context

Abstract 939: The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells

Abstract Background: B-cell lymphoma 2 (Bcl-2) is an oncoprotein that promotes tumor cell survival and drug resistance. Elevated Bcl-2 expression in numerous human cancers has made it an ideal drug target. Venetoclax, the first FDA approved Bcl-2 inhibitor, is indicated for hematologic malignancies. Accompanying its promising clinical results, however, is the emergence of venetoclax resistance. A recent study found that AML patients who had relapsed from frontline venetoclax-based treatment were refractory to salvage therapy and had a median survival of less than 3 months. Thus, novel treatment approaches for such patients are urgently needed. BP1002, a non-toxic neutral dioleoylphosphatidylcholine liposome incorporating a nuclease-resistant P-ethoxy antisense oligonucleotide (oligo), was developed to target Bcl-2 mRNA. The safety and efficacy of BP1002 is being investigated in patients with advanced lymphoid malignancies. Venetoclax in combination with decitabine is an approved treatment for elderly AML patients. Here we determine the effects of BP1002 plus decitabine in venetoclax-resistant cells. Methods: Cell lines: Venetoclax-resistant hematologic cell lines, MV-4-11- R, SU-DHL-2-R, SU-DHL-6-R, were established from the parental cells by exposing the cells to increasing doses of venetoclax for 10-12 weeks.BP1002: A Bcl-2 antisense oligo, which targets the translation initiation site of human Bcl-2 mRNA, was incorporated in BP1002. Combination study: Parental and venetoclax-resistant cells were treated with decitabine for 2 days followed by the addition of BP1002 for another 4 days of incubation. Resazurin-based cell viability assays and Western blots were conducted to determine the inhibitory effects of BP1002 in parental and resistant cells. Results: The IC50 values of venetoclax in resistant cells were 3 to 40 times higher than those in parental cells. MV-4-11-R, SU-DHL-6-R, SU-DHL-2-R cells are resistant up to 0.1 μM, 0.8 μM, and 7 μM venetoclax, respectively. Compared to parental cells, venetoclax-resistant cells displayed 2- to 3-fold elevated levels of Bcl-2 protein and other anti-apoptotic proteins such as Mcl-1. BP1002 in combination with decitabine decreased the viability of parental cells. BP1002 reduced Bcl-2 levels in venetoclax-resistant cells. Also, the BP1002 plus decitabine combination was more potent than the venetoclax plus decitabine combination in reducing the viability of the resistant cells, by about 50-70%. Conclusions: Our data indicate that, despite expressing higher levels of anti-apoptotic proteins, venetoclax-resistant cells are sensitive to the BP1002 plus decitabine combination. These promising preclinical results suggest that the BP1002 plus decitabine combination is a potential treatment for patients who have relapsed from frontline venetoclax-based therapies. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 939.

MYC suppresses STING-dependent innate immunity by transcriptionally upregulating DNMT1 in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks definite treatment targets. Tumor immune microenvironment (TIME) heterogeneity has a profound impact on the immunotherapy response....

Abstract 1049: Optimizing the treatment schedule of selinexor in combination with decitabine in AML

Abstract Background: Decitabine (DEC) is a hypomethylating agent approved to treat acute myeloid leukemia (AML) that can reactivate tumor suppressor genes that are epigenetically silenced by DNA methylation. Selinexor (SEL) is a selective inhibitor of nuclear export that inhibits exportin 1 (XPO1/CRM1) from shuttling cargoes, including tumor suppressor proteins and oncogene mRNAs bound to eIF4e, from the nucleus to the cytoplasm. DEC priming has been previously found to increase the antileukemic effects of SEL in AML in vitro and in vivo, through DEC-mediated upregulation of tumor suppressor proteins such as CDKN1A (p21) and FOXO3A, which are XPO1 cargos localized in the nucleus after SEL treatment. The goal of this study was to determine the optimal dosing schedule for combination treatment of SEL and DEC in AML. Methods: The efficacy of concomitant and sequential drug treatment using DEC and SEL was evaluated in the subcutaneous implanted MV4-11 xenograft models of AML in NOD-SCID mice. Tumor bearing mice were treated with vehicle, SEL once weekly at 15 mg/kg, DEC at 0.4 mg/kg (M-F, week 1 only) and combinations of DEC with SEL 15 mg/kg on Mondays only (DEC + SEL M, concomitant) or Fridays only (DEC + SEL F, sequential) starting from week 1. In the follow up experiment mice were treated similarly except for revised combination group schedules, SEL was administered once weekly starting either from Friday of week 1 (sequential A) or Monday of week 2 (sequential B). Tumor volume and body weight of mice were monitored for five weeks of treatment, and tumors were collected at the end of the study for immunohistochemistry (IHC) analysis. Results: DEC and SEL effectively inhibited tumor growth as single agents. Concomitant administration of SEL and DEC did not enhance the anti-tumor effect, with no significant difference among the tumor growth inhibition (TGI) rate for mice treated with SEL alone (62.7%), DEC alone (60.0%) and the concomitant combination (DEC + SEL M, 62.6%) (p&amp;gt;0.05). Whereas sequential administration of SEL after DEC priming (DEC + SEL F) enhanced the anti-tumor effects, as TGI (81.7%) was significantly higher than each single agent alone (p&amp;lt;0.05). In the follow up experiment, TGI for the two sequential DEC and SEL combination groups was similar (86.19% for sequential schedule A and 86.17% for sequential schedule B, p&amp;gt;0.05). Conclusions: The introduction of SEL post priming with DEC showed greater TGI than when SEL and DEC were dosed concomitantly. A delay of 4-7 days between the first dose of SEL after DEC priming demonstrated better antileukemic effect than concomitant dosing. This is relevant to potential future clinical trials because separation of dosing DEC and SEL has the potential to increase tolerability of the combination without affecting antileukemic activity. Citation Format: Leah F. Henegar, Hua Chang, Christopher J. Walker, Trinayan Kashyap, Nicholas Fredette, Kathleen Martyn, Sharon Shacham, Yosef Landesman. Optimizing the treatment schedule of selinexor in combination with decitabine in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1049.

Abstract 1113: Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models

Abstract Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy and relapse within short time, leaving no further therapeutic option than bone marrow transplant, when feasible. This highlights the need to identify new first-line therapies for high-risk patients.We explored the potential of upfront drug combinations to prevent the rise of resistant disease, in vitro and in vivo. Crizotinib was combined with CHOP chemotherapy, an epigenetic drug (decitabine), another targeted inhibitor (trametinib- MEK inhibitor), or a second-generation ALK inhibitor. ALK+ ALCL cells were kept in culture for ~100 days in the presence of different single or combined drugs. We found that in most cases, combined treatments were more effective than single agents in the long-term control of cells expansion. Upon further functional characterization using quantitative PCR and western blot, we found that combination treatments upregulated cell cycle inhibitors and pro-apoptotic genes as well as modulated survival factors to induce deeper inhibition of oncogenic signalling and higher rates of apoptosis. Crizotinib+decitabine combination treatment promoted downregulation of DNMT1 and increase of TGFB1, both of which indirectly affect cell cycle progression. In murine model, crizotinib+decitabine significantly delayed tumour growth, compared to single treatments. Our results indicate that combining a selective drug with a less specific anticancer agent, which allows unspecific killing of resistant clones that might potentially arise, would be beneficial.In conclusion, our data suggest that a polypharmacology approach combining ALK inhibition with other agents could be a valuable therapeutic option for ALK+ ALCL patients, to prevent relapses and improve outcomes. Citation Format: Geeta G. Sharma, Giulia Arosio, Matteo Villa, Mario Mauri, Marina Zappa, Vera Magistroni, Monica Ceccon, Sara Redaelli, Ilaria Crespiatico, Diletta Fontana, Cristina Mastini, Anna Garbin, Federica Lovisa, Lara Mussolin, Rocco Piazza, Carlo Gambacorrti-Passerini, Luca Mologni. Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1113.