IntroductionThe main purpose of the current study was to investigate the antitumor effects of 5-methoxypsoralen in U87MG human glioma cells along with studying its effects on cell cycle progression, autophagy and the PI3K/Akt signaling pathway.Material and methodsThe cytotoxic effects of the drug were demonstrated by the MTS cell viability assay while its effects on cellular morphology associated with cell apoptosis were evaluated by phase contrast and fluorescence microscopic techniques. Effects of 5-methoxypsoralen on the cell cycle were studied by flow cytometry while effects on PI3K/Akt proteins were evaluated by western blot assay.ResultsThe results indicate that 5-methoxypsoralen led to time-dependent as well as dose-dependent inhibitory effects on U-87MG human glioma cells. 5-Methoxypsoralen led to a substantial decrease of cell count along with distorted cell morphology. The molecule also led to DNA ladder formation which increased with increasing doses of 5-methoxypsoralen. 5-methoxypsoralen also led to dose-dependent G2/M phase cell cycle arrest. 5-Methoxypsoralen-treated cells also exhibited altered cell ultrastructure with the appearance of autophagic vacuoles and the number of these vacuoles increased with increasing drug dose.ConclusionsIn brief, the results indicate that 5-methoxypsoralen exerted potent anticancer and apoptotic effects in U-87MG human glioma cells along with inducing cell cycle arrest, autophagy and m-TOR/PI3K/Akt signaling pathway inhibition.

Background Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in treating a variety of blood cancers, such as CD19 CAR-T for B-cell malignancies and BCMA CAR-T for myeloid myeloma (MM). However, similar progress has yet to be achieved with relapsed and refractory acute myeloid leukemia (R/R AML), primarily due to the heterogeneous nature of AML, making it difficult to find an ideal CAR-T target. Previous efforts have targeted single CD33, CD123, LeY, NKG2D, or CD70 receptors, but the overall response rate is much lower compared to CD19 CAR-T. Improved clinical outcome was recently reported with a dual-receptor CAR-T (CD33 and CLL1), reaching ~80% CR for R/R AML patients. However, CD33 is also expressed in normal hematopoietic stem cells (HSC) and patients need allogeneic hematopoietic stem cell transplantation (HSCT) following CAR-T therapy. Notably, AML mainly affects the elder population, and patients more than 65 years old are usually not suitable for HSCT. To address these challenges, we aim to find an effective target for AML without the need for the HSCT. In our study, CLL1 is chosen as a promising target as it is not expressed on normal HSCs, but highly expressed on AML blasts cells and leukemia stem cells (LSCs), which is a small population that plays an important role in disease progression and relapse. Here we report the results from a Phase I clinical trial to evaluate the toxicity and efficacy of the CLL1 CAR-T in the treatment of pediatric R/R AML. Methods We have generated a 2nd generation of CLL1 CAR-T, the extracellular scFv was derived from a murine CLL1 monoclonal antibody, which was generated by hybridoma technology. Autologous CAR-T cells were manufactured in a cGMP facility. Between Oct 2019 and Mar 2020, 3 pediatric R/R AML patients were infused. CAR-T cells were given by a dose at 0.2-1x106/kg with a single dose. Results Of the 3 patients infused, cytokine release syndrome (CRS) occurred in 3 patients (2 grade Ⅰ, 1grade II), no neurotoxicity occurred. All patients suffered pancytopenia, granulocytopenia and monocytopenia. All the adverse effects were resolved after treatment. Patient 1 is a refractory AML patient with more than 95% of AML blasts being CLL1 positive. The infused CAR-T cells showed typical clonal expansion peaking at Day 8. The patient reached CR/MRD- when evaluated at Day 21 post infusion. The patient received HSCT at Day 35, and remained CR/MRD- till now (Jul 2020, 8 months post CAR-T infusion). Patient 2 is a relapsed patient after HSCT and showed severe bone marrow necrosis (BMN). Therefore, we only generated a dose 0.35-1x106/kg CAR-T cells. The patient reached CR/MRD- when evaluated at Day 14 post infusion. The patient went through a second HSCT at Day 38. However, the patient passed away due to GVHD two months after the second HSCT. Patient 3 is a refractory AML patient with about 85% of AML blasts being CLL1 positive. The patient reached CR/MRD+ (1%) when evaluated at Day 14 post infusion. However, AML blast increased to 11% when evaluated at Day 30, a majority of them were CLL1 negative. The patient received Azacitidine treatment at Day 30-34, and AML blast decreased to 2.8% at Day 45. Notably this patient was refractory to Azacitidine treatment previously, suggesting that CLL1 CAR-T could eradicate LSCs, making the left AML tumor cells sensitive to chemotherapy. This patient received HSCT at Day 75, and remained CR/MRD- till now (Jul 2020, 7 months post CAR-T infusion). Conclusion Our study suggested that CLL1 CAR-T is a therapy with high efficacy and manageable toxicity in R/R AML patients. All patients in this study could reach CR within one month and only experienced 1-2 grade CRS. For patients with CLL1 negative AML blast, the CLL1- cells may take over after CLL1 CAR-T therapy. Combination with chemotherapy like Azacitidine may help patients reach complete response. Figure Disclosures No relevant conflicts of interest to declare.

Background: Unrelated cord blood (UCB) transplant (UCBT) is not recommended in patients with thalassemia major (TM) so far. Post-transplant (PT) Cyclophosphamide (PTCy) with long pre-transplant immunosuppression therapy have improved haploidentical peripheral blood (PB) stem cell transplant (haplo-SCT) survival in TM patients but with 2/31 primary rejection. So, we designed a novel dual transplantation of UCBT following haplo-SCTwith PTCy(NF-14-TM-CT protocol). Aim:To improve results of haplo-SCT and UCBT in patients with TM. Patients and method: NF-14-TM -CT protocol was termed as double-insurance dual transplantsincluding a haplo-SCT and an UCBT, in which conditioning regimen consisted of ATG (at -10 to -8 day), Cy (-7), Fludarabine (-6 to -2), Busulfan (-6 to -4) Thiotepa (-3), haplo-PB (0), PTCy (+3, +4) and UCB (+6). PTCy serve as GVHD prophylaxes after haplo-SCT and as conditioning before UCBT. In total 131 patients with TM from three pediatric center in China received NF-14-TM-CT protocolfrom June, 2014 to April, 2019, with a median follow-up of 13 (2-59) months and a median age of 8 (3.5-17) years. Results Final haplo-PB engrafted(group1)in 76 patients with mean PBSC-MNCof 22.49 (±5.36) x108/kgand UCB nuclear cells (NC) of 5.95 (±3.39) x107/kg and final UCB engrafted (group 2) in 55 patients with mean PBSC-MNC of 21.78 (±5.68) x108/kg and mean UCB-NC of. 5.43 (±2.32) x107/kg. The 4-year overall survival (OS), thalassemia-free survival (TFS), graft rejection (GR), and transplant related mortality (TRM) were 97.6%, 96.0%, 1.5%, and 2.4%, respectively (Fig. A), in total. The corresponding rates for group 1 were 98.3%, 96.9%, 1.7% and 1.8% and for group 2 were 95.5%, 93.8%, 4.5% and 1.4%, respectively. No statistic significant difference was found in OS, TFS, GR and TRM, respectively, when comparing group 1 with group 2 (Fig. B. C, D, E).The incidence of grade II-IV acute GVHD, III-IV acute GVHD, mild chronic GVHD, moderate/severe chronic GVHD, VOD, PT cystitisand PT hemolysis of the entire cohort was 16.8%, 6.87%, 9.92%, 1.52%, 4.60%, 31.3% and 14.5, respectively. Summary:Current study proved that the novel CT improved the results of haplo-SCT and UCBT in patients with TM. Disclosures Wing: Miltenyi Biotec: Employment.

10000 Background: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable success in treating a variety of blood cancers, such as CD19 CAR-T for B-cell malignancies and BCMA CAR-T for myeloid myeloma (MM). However, similar achievement has yet to be replicated in patients with relapsed and refractory acute myeloid leukemia (R/R AML), primarily due to the AML heterogeneity, making it difficult to find an ideal CAR-T target. Previous efforts have targeted single CD33, CD123, LeY, NKG2D, or CD70 receptors, but the overall response rate is very disappointed. To address these challenges, we aim to find an effective target for AML without the need for the hematopoietic stem cells transplant (HSCT). In our study, CLL1 is chosen as a promising target as it is not expressed on normal HSCs, but highly expressed on AML blasts cells and leukemia stem cells (LSCs). Here we report the interim analysis from a Phase I clinical trial using anti-CLL1 based CAR-T cells to treat children with R/R AML. The primary and secondary objectives were to evaluate the safety and anti-AML responses, respectively, with long-term prognosis within those patients who did not receive allogeneic HSCT (allo-HSCT) as an additional objective. Methods: We have generated a 2nd generation of CLL1 CAR-T, the extracellular scFv was derived from a murine CLL1 monoclonal antibody, which was generated by hybridoma technology. Autologous CAR-T cells were manufactured in a cGMP facility. Between Oct 2019 and Jan 2021, 11 pediatric R/R AML patients were infused. CLL1 or CLL1-CD33 dual CAR-T cells were given by a dose at 0.3-1x106/kg with a single dose after lymphodepleting conditioning with cyclophosphamide/fludarabine(Cy/Flu). Results: Of the 11 patients infused, Grade 3-4 hematologic adverse events were observed before and during CAR-T cell infusion, and no dose-limiting toxicities were observed. Meanwhile, grade 1-3 cytokine release syndrome was observed but without any lethal events. All the adverse effects were resolved after guideline-directed intervention. Anti-CLL1 CAR-T cells efficiently expanded in vivo, the median expansion peaking time was at Day 8. For these 11 R/R-AML patients, 10 patients completely responded to anti-CLL1 based CAR-T cell therapy, with CLL1 positive AML blast eliminated within one month. Among the responded 10 patients, 5 patients reached CR/MRD-, 3 patients reached CR/MRD+, 1 patient reached PR and 1 patient showed SD, with only CLL1 negative AML cells. Conclusions: Our study demonstrated that 10/11 patients responded to CLL1 CAR-T cell therapy within one month. For patients showing MRD+ with CLL1 negative AML blast, chemotherapy like Azacitidine, and combined with HSCT may help those patients to reach complete response. These initial results suggested that anti-CLL1 base CAR-T cells can be a well-tolerated and candidate option for treating children with R/R-AML. Clinical trial information: ChiCTR1900027684.