ALBERT

Description: Abstract 1360 Introduction: The tyrosine kinase inhibitor (TKI) imatinib is used as the first-line therapy for newly diagnosed chronic myeloid leukemia (CML). However, some patients fail to respond or become intolerant to imatinib. Nilotinib is a second-generation TKI with higher selectivity and more potent inhibitory effects on BCR-ABL than imatinib. Several studies have shown hematologic and cytogenetic responses to nilotinib in patients with imatinib-resistant or intolerant CML. Purpose: To investigate the safety and efficacy of nilotinib for patients with imatinib-resistant or intolerant, chronic (CP)- or accelerated (AP)-phase CML from the East Japan CML Study Group (EJCML) trial by evaluating molecular responses in terms of the BCR-ABL1 mutational status and plasma trough concentration of nilotinib. Methods: In this multicenter phase II clinical trial, nilotinib (400 mg bid) was administered orally for one year and the molecular responses were monitored by means of the international scale of quantitative PCR (IS-PCR). BCR-ABL1 mutations were analyzed by direct sequencing at the baseline and 12 months or at the time of the event for discontinuation of the treatment (i.e., progressive disease, insufficient effects, or severe adverse events). The plasma trough concentration of nilotinib was measured by high-performance liquid chromatography 3 months after nilotinib administration. Results: From March 2009 through February 2011, 51 patients were registered in this study, and data of 49 patients whose molecular responses were evaluated by the IS-PCR were analyzed (imatinib-resistant CML = 33, imatinib-intolerant CML = 16; CP CML = 46, AP CML = 3). The median follow-up period was 12.0 months (range = 0.1–13.3 months). At 6 and 12 months, the major molecular response (MMR; ≤0.1% IS) rates were 52.5% and 67.6%, respectively, and the complete cytogenetic response (CCyR)-equivalent (≤1.0% IS) rates were 75.0% and 85.3%, respectively. Five types of BCR-ABL1 mutations (M244V, F317L, N358D, F359V, and E459K) were detected in 6 patients (12.2%) at the baseline, but the M244V, N358D, and E459K mutations disappeared after the nilotinib treatment. Acquired BCR-ABL1 mutations (Y253H, I418V, and exon 8/9 35bp insertion) were detected in 3 patients (8.6%) at 12 months or at the time of the event; these patients did not achieve a CCyR or an MMR. No patients showed an acquired mutation of T315I. Most patients except 11 subjects (22.4%) still received the treatment. The reasons for discontinuation were progressive disease in one patient with an F317L mutation, insufficient effects in one patient without any mutation, and adverse events in 9 patients (thrombocytopenia in 5 patients, hyperbilirubinemia in 2 patients, headache in one patient, and heart disease in one patient). Among 30 patients without BCR-ABL1 mutations, the plasma trough concentration of nilotinib was significantly higher in 21 patients with an MMR than in those without an MMR by 12 months (median = 1255.1 ng/mL vs. 372.8 ng/mL, P = 0.0012 by Mann–Whitney U-test; see the figure). The concentration of 761 ng/mL was significantly associated with an MMR by 12 months in a receiver-operating characteristic (ROC) curve analysis of the best sensitivity (76.2%) and specificity (77.8%). Conclusion: The patients with imatinib-resistant or intolerant, CP or AP CML, even those having BCR-ABL1 mutations M244V, N358D, and E459K, achieved an MMR by 12 months of nilotinib treatment. The plasma trough concentration of the drug was related to the MMR by 12 months, and the plasma threshold of nilotinib should be set above 761 ng/mL. These findings suggest that nilotinib shows good efficacy and tolerability in Japanese patients with imatinib-resistant or intolerant, CP or AP CML. (ClicalTrials.gov, UMIN ID 000002201) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1684 Imatinib masylate (IM) induces sustained molecular remissions in patients with chronic myeloid leukemia (CML) but a life-long therapy is required for most of these patients. In STIM study, Mahon et al. reported that among patients with a complete molecular response (CMR) lasting at least two years, the CMR was sustained in 41% after discontinuation of IM. German study group showed that treatment with Interferon-alpha (IFN) enables IM discontinuation in most patients after prior IM/IFN combination therapy. We previously reported that CD8+ memory T cells showed significant predominance over naive T cells in the patients with sustained therapy-free major molecular response (MMR), all of that had previously received IFN [1]. We have been performing a phase 2 study of treatment discontinuation after the drug change from IM to IFN (Japanese Imatinib Stop And Interferon Study; JISAS). Since the aim of this study is to confirm the finding that IFN is able to maintain MMR induced by IM after its discontinuation, we present here the result of the interim analysis. Patients Patients with a confirmed diagnosis of CML in 1st chronic phase (CP1) as well as in CMR (undetectable BCR-ABL transcript) following over 2 years of MMR on IM were enrolled in this pilot study after obtaining the written informed consent. All the eligible patients were recruited from September 2009 to December 2011 whether or not any therapeutic drug had been given before IM. Evaluation and response criteria Since a conversion factor for International scale was not available in Japan until recently, for entry to the study, BCR-ABL transcripts at a level equal to or below 100 copy/mg RNA in a real-time quantitative-polymerase chain reaction (RQ-PCR) assay or equal to or below 50 copy/assay in a highly sensitive transcription-mediated amplification (TMA) method were defined as MMR. CMR was defined as detection of no BCR-ABL transcript in RQ-PCR assay, nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay, or TMA. During the study, molecular response was assessed at the baseline and every month thereafter, by determining the BCR-ABL to ABL mRNA transcript ratio isolated from the peripheral blood using RQ-PCR and RT-PCR. The BCR-ABL to ABL transcript ratio at a level below 0.001 was defined as MMR and no detectable BCR-ABL transcript in RT-PCR was defined as CMR. Molecular relapse defined as a loss of MMR was taken into account if confirmed in 2 successive assessments. Study design and treatment Administration of IFN is started at a dose of 3 million units 2–5 times per week within 4 weeks after IM discontinuation. In case of molecular relapse, IM was resumed at 400 mg daily. Statistical analysis Non-parametric values or numbers were compared between the two groups using the Mann-Whitney test. Relapse-free survival was estimated using the Kaplan-Meier method. Results Fifteen patients were enrolled from September 2009 to December 2011. Median age was 50 years (range, 28–67 years) and male to female ratio was 1.5. Sokal score at the diagnosis was low in 13 patients and high in 2 patients. Four patients had been treated before IM. Previous therapies comprised IFN in all these patients, including 1 patient who relapsed after allogeneic stem cell transplantation (SCT). The clinical stage of this patient was amended to be accelerated phase at the time of diagnosis. Two other patients withdrew the consent. Excluding these 3 patients, the remaining 12 patients with low risk of Sokal score were analyzed. The median follow-up period is 23 months (range: 6–27 months). Three patients lost MMR (1, 3, and 6 months, respectively) and other 9 maintained CMR. Molecular relapse-free survival is 74%. The sustained CMR patients had the significantly longer CMR period on IM (median 31 months, range 26–79 months) compared with relapsed patients (0, 9, and 14 months, respectively; p=0.0142). There was no difference between the relapsed and the sustained CMR patients in the duration of IM treatment. All the relapsed patients achieved MMR after 2, 4, and 5 months of IM resumption, respectively. In conclusion, IFN monotherapy is a promising option for sustained molecular response after IM discontinuation in CML patients with CMR. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2442 Cyclodextrins (CyDs) are cyclic oligosaccharides that can remove cholesterol from cell membranes and thereby affect receptor function, and are widely used in the pharmaceutical field because of those abilities to improve solubility, dissolution rate and bioavailability of the drugs. A number of studies have demonstrated that methyl-β- cyclodextrin (MβCD) can damage tumor cells and induce cell death. Very recently, Yan et al reported, on the basis of in vitro experiments, that MβCD induces apoptosis of chronic myeloid leukemia (CML) cells and have synergistic anti-leukemic effect combined with imatinib. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is clinically used as a pharmaceutical excipient, which has been successfully applied to poorly water-soluble drugs. Recently HP-β-CyD has been approved for the treatment of Niemann-Pick type C disease, a rare lysosomal lipid storage disorder. In the present study, we examined the antiproliferative effect of HP-β-CyD on the in vitro growth of leukemic cell lines and in vivo model using mice transplanted with leukemic cell line. First in vitro proliferation was assessed using the modified MTT assay. The human Ph+ leukemic cell line BV173 and BaF3 cells expressing p190 wild type BCR-ABL (hereafter BaF3/BCR-ABL) were used for evaluation. The growth of BV173 and BaF3/BCR-ABL cells were similarly inhibited by HP-β-CyD in a time- and dose-dependent manner with IC50 value of 4.68 ± 0.98 and 6.01 ± 1.04 mM, respectively. In contrast, IC50 value for hepatocytes was 18.65 ± 4.84 mM, suggesting some therapeutic window between normal cells and CML cells. We next determined if the inhibition of leukemic cell growth by HP-β-CyD was associated with the induction of apoptosis. BaF3/BCR-ABL and BV173 cells were exposed to HP-β-CyD for 12, 24, 48 hours at concentrations of 5, 10, 15 and 25mM. Assessment of apoptosis by 7-AAD/Annexin V double staining revealed that HP-β-CyD induced apoptosis in both cell lines in a time- and dose-dependent manner. The toxicity of HP-β-CyD on normal hematopoietic progenitors was also examined. The susceptibility of normal hematopoieic progenitors was investigated by colony-forming units (CFU-C) assay. When normal progenitors were treated with 5, 15 or 25 mM HP-β-CyD, the percentage of colonies was 92.7 ± 8.6 %, 83.8 ± 23.5 % and 52.4 ± 9.7 % of control, respectively. These results also indicate that HP-β-CyD may not induce bone marrow suppression up to 15mM. Because in vitro assay showed significant effects against leukemia cell growth, we also investigated the in vivo efficacy of HP-β-CyD. Six-week-old nude mice were injected with 1×106 BaF3/BCR-ABL cells, and were intraperioneally treated with 200 μl of either vehicle, 50mM HP-β-CyD or 150mM HP-β-CyD twice a day (BID) for 20 days from 3days after transplantation. The vehicle-treated mice died of a condition resembling acute leukemia by 29 days after transplantation; HP-β-CyD- treated mice survived more than 40 days, significantly improved the survival (50mM: P=0.003, 150mM: P=0.001, respectively) compared with control mice (Figure 1). These results clearly demonstrate that HP-β-CyD itself has a certain level of anti-leukemic potential. Though further investigations are required to elucidate the mechanisms underlying the antiproliferative function of HP-β-CyD, we should take notice of additional effect when the evaluation of drug efficacy is performed for anti-cancer agents complexed with HP-β-CyD. Figure 1. Administration of HP-β-CyD prolonged the survival in mice model of BCR-ABL-induced leukemia. Nude mice were injected with 1×106 BaF3 cells expressing p190 BCR-ABL. These mice were treated with vehicle or HP-β-CyD (50 or 150mM) for 20days from 3 days after transplantation. Figure 1. Administration of HP-β-CyD prolonged the survival in mice model of BCR-ABL-induced leukemia. Nude mice were injected with 1×106 BaF3 cells expressing p190 BCR-ABL. These mice were treated with vehicle or HP-β-CyD (50 or 150mM) for 20days from 3 days after transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2773 Development of the second-generation ABL tyrosine kinase inhibitor (TKI) dasatinib in CML aimed at overcoming resistance to imatinib, to eliminate persistent residual disease and thus prevent recurrence of active leukemia after TKI discontinuation in chronic myeloid leukemia (CML). Hypoxia has recently been reported as an essential component of the leukemia BM microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito et al, PlosOne 2011). In this study, we investigated the anti-CML efficacy and molecular mechanisms of action of dasatinib under hypoxic conditions. We developed a hypoxia-adopted subclone of the KBM5 CML cell line (KMB5-HA), which was selected under 1.0% oxygen conditions, and an imatinib-resistant KBM5 subline bearing the T315I mutation (KBM5-T315I). KBM5-HA cells cultured under hypoxia accumulated in G0/G1 and exhibited moderate spontaneous apoptosis compared to KBM5 parental cells in normoxia (sub G1 %: KBM5 3.8±0.8, KBM5-HA 8.7±0.9; p=0.01; proportion of cells in G0/G1: KBM5 36.5±1.1%, KBM5-HA 52.4±6.6%; p=0.02, PI analysis). These cells displayed higher sensitivity to dasatinib than parental KBM cells (IC50; 1.3 nM for KBM5, 0.3 nM for KBM5-HA, at 48hrs by MTT). Low-dose dasatinib (0.5nM) which failed to cause inhibition of proliferation in parental KBM5 cells, caused significant apoptosis induction with cell cycle arrest in KBM-HA cells (sub G1 %: control 8.7±0.9, datatinib 49.1±16.9; p=0.02, G0/G1 %: control 52.4±6.4%, dasatinib 74.1±3.5%; p=0.01). In KBM5-T315I cells, dasatinib induced more prominent cell growth inhibition under hypoxia compared to normoxia (IC50 at 48hrs: normoxia 16.2 nM, hypoxia 3.7 nM). Treatment with 5nM dasatinib, which did not affect KBM5-T315I cell growth under normoxia, induced significant apoptotic effects under hypoxia (sub G1 %: control 6.9±0.9, dasatinib 20.3±3.3; p=0.05, G0/G1 %: control 57.3±5.8, dasatinib 67.4±8.9; p=0.41). We next investigated dasatinib-induced changes of Stat-5 and ERK activation in CML cells by immunoblotting Treatment with 0.5 nM dasatinib resulted in marked down-regulation of phosphorylated (p-) Stat-5 and p-ERK in both, KBM5 and KBM5-T315I cells. In KBM5-HA cells, however, no baseline expression of p-Stat-5 or p-ERK was detected. These results suggest that in KBM5-HA cells dasatinib induces apoptosis and cell cycle arrest via Stat-5 or ERK-independent pathways. To investigate the candidate signaling factors responsible for dasatinib effects on KBM-HA, we performed the proteomic analysis utilizing proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems) coupled with two-dimensional-liquid chromatography-tandem mass spectrometry. A total of 1,234 proteins were detected, and 296 proteins were found to be significantly up-regulated in response to dasatinib treatment in KBM5-HA cells. Among the up-regulated proteins, we found 39 proteins involved in apoptosis induction including Cytochrome C and Cytochrome C oxydase subunits (p

Description: Abstract 2805 Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3'UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS. We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to 〉90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p

Description: Abstract 2459 Prognosis of chronic myeloid leukemia (CML) has been dramatically improved by the induction of tyrosine kinase inhibitors (TKIs) as a first-line treatment. However, cure is still difficult because of the existence of leukemic stem cell (LSC). Zoledronic acid (ZOL) is a 3rd-generation bisphosphonate that inhibits bone matrix resorption of osteoclasts (OCs), and inhibit the proliferation of several tumor cells by preventing posttranslational prenylation of Ras-related proteins. We have previously reported that ZOL possess anti-leukemic effect that augments synergistically the activity of imatinib mesylate (Kuroda, et al. Blood 2003), and OCs maintain leukemic cells in a quiescent state under newly developed co-culture system (Yokota, et al. Leuk Res 2010). Thus, we hypothesized that the treatment of ZOL combined with nilotinib (Nilo) may be effective to eliminate dormant leukemic cell population, called LSCs, by attacking the OCs. To test this hypothesis, we examined the combined effect of Nilo and ZOL on the human CML cell line BV173 and BaF3 cells expressing p190 wild type BCR-ABL (BaF3/BCR-ABL) in vitro. Firstly, modified MTT assay was performed with ordinary condition (without any other co-cultured cells), resulting that ZOL augmented synergistically in vitro effects of Nilo both on cell lines (combination index 〈 1 at Fa=0.5) (Figure 1). Next we investigated the anti-leukemic effects of Nilo and ZOL using our developed co-culture system including osteoblasts (OBs), OCs and bone slices (Yokota, et al. Leuk Res 2010). 104 BaF3/BCR-ABL cells were applied to transwell chambers in each well of the co-culture plate (thus, BaF3/BCR-ABL cells did not attach directly to OBs, OCs and bones). Next day, each well was exposed to saline, Nilo 10nM, ZOL 10uM or Nilo 10nM plus ZOL 10uM. 48 hours after treatment, the viable cell number of BaF3/BCR-ABL was counted by trypan blue staining. Cell numbers were as follows; control: 55.79×104/well, Nilo only: 36.17×104/well, ZOL only: 64.46×104/well, Nilo plus ZOL: 39.42×104/well, respectively (Figure 2). These results suggested that combination effect of Nilo plus ZOL in vitro was canceled in the co-culture system. In the same condition, 7-AAD/Annexin V double staining was also performed. The apoptotic rates were as follows; control: 38.05 ± 2.36 %, Nilo only: 29.49 ± 2.59 %, ZOL only: 37.67 ± 1.39 %, Nilo plus ZOL: 29.66 ± 5.67 %. The results indicated that co-culture system of mimic the bone marrow microenvironment changed the drug effect on BaF3/BCR-ABL to resistance. To reveal the mechanisms of this phenomenon, TGF-ƒÀ1 concentration in the co-culture supernatants was measured by ELISA. In the previous report, when BaF3/BCR-ABL were co-cultured with OBs, OCs and bone slices, their proliferation was significantly suppressed and TGF-ƒÀ1 was significantly higher in supernatants from the co-culture system consisted of OB+OC+bone than control. In the present study, TGF-ƒÀ1 ratios (TGF-ƒÀ1 concentration in each well administrated with each agent/that in the medium without any cells) were almost same among wells treated with each agent. These results suggested that TGF-ƒÀ1 concentrations maintained for a while even after OCs were destroyed by ZOL and the remained TGF-ƒÀ1 affected the effects of anti-cancer agents on the proliferation of CML cells. In conclusion, the present study suggested that the evaluation of combined effects in ordinary culture system without any other cells which consist of microenvironment might not be correctly reflected in more physiological system. Therefore, it may be important to utilize the co-culture system used here to evaluate the effects of anti-cancer agents. Figure 1. Combination effects of Nilo and ZOL evaluated in ordinary condition. To examine the combined effects of Nilo with ZOL on (A) BaF3/BCR-ABL and (B) BC173, modified MTT assay was performed with ordinary condition (without any other co-cultured cells). The fraction affected (Fa) (i.e. Fa of 0.5 would equal 50% viable cells) and the combination index (CI) were calculated with CalcuSyn (Biosoft). Figure 1. Combination effects of Nilo and ZOL evaluated in ordinary condition. . / To examine the combined effects of Nilo with ZOL on (A) BaF3/BCR-ABL and (B) BC173, modified MTT assay was performed with ordinary condition (without any other co-cultured cells). The fraction affected (Fa) (i.e. Fa of 0.5 would equal 50% viable cells) and the combination index (CI) were calculated with CalcuSyn (Biosoft). Figure 2. Combination effects of Nilo and ZOL evaluated in co-culture system. Living cell numbers which were treated with saline (no treat), Nilo, ZOL and Nilo plus ZOL (N+Z) either in control culture system without any co-cultured cells (white bar) or co-culture system (black bar) were counted by trypan blue assay. Co-culture system consisted of osteoblasts, osteoclasts and bone slices. Figure 2. Combination effects of Nilo and ZOL evaluated in co-culture system. . / Living cell numbers which were treated with saline (no treat), Nilo, ZOL and Nilo plus ZOL (N+Z) either in control culture system without any co-cultured cells (white bar) or co-culture system (black bar) were counted by trypan blue assay. Co-culture system consisted of osteoblasts, osteoclasts and bone slices. Disclosures: No relevant conflicts of interest to declare.

Description: EBV-associated T/NK–cell lymphoproliferative disease (T/NK-LPD) is defined as a systemic illness characterized by clonal proliferation of EBV-infected T or NK cells. We prospectively enrolled 108 nonimmunocompromised patients with this disease (50 men and 58 women; median onset age, 8 years; age range, 1-50 years) evidenced by expansion of EBV+ T/NK cells in the peripheral blood; these were of the T-cell type in 64 cases and of the NK-cell type in 44, and were clinically categorized into 4 groups: 80 cases of chronic active EBV disease, 15 of EBV-associated hemophagocytic lymphohistiocytosis, 9 of severe mosquito bite allergy, and 4 of hydroa vacciniforme. These clinical profiles were closely linked with the EBV+ cell immunophenotypes. In a median follow-up period of 46 months, 47 patients (44%) died of severe organ complications. During the follow-up, 13 patients developed overt lymphoma or leukemia characterized by extranodal NK/T-cell lymphoma and aggressive NK-cell leukemia. Fifty-nine received hematopoietic stem cell transplantation, 66% of whom survived. Age at onset of disease (≥ 8 years) and liver dysfunction were risk factors for mortality, whereas patients who received transplantation had a better prognosis. These data depict clinical characteristics of systemic EBV+ T/NK-LPD and provide insight into the diagnostic and therapeutic approaches for distinct disease.

Description: Abstract 1901 Majority of AML blasts express CD38, while human hematopoietic stem cells express CD34 but not CD38. Accordingly, we focused on CD38 for a therapeutic target and developed a cellular immunotherapy by using T cells bearing anti-CD38-chimeric antigen receptor (CAR). We recently reported T cells with anti-CD38-CAR efficiently eliminated B-cell lymphoma cells expressing CD38 in vitro and in vivo. However, intensity of CD38 in lymphoma cells is much higher than that in AML cells. Additionally, leukemic stem cells(LSCs) phenotypically express CD34+CD38−. Thus, to fully employ anti-CD38-CAR against AML blasts including LSCs, intensity of CD38 expression need to be raised for the clinical application. All-trans retinoic acid (ATRA) is widely used for treatment of patients with acute promyelocytic leukemia (APL). Interestingly, it has been reported that ATRA enhances CD38 expression on the surface of AML cells. In this study, we investigated whether human peripheral T cells retrovirally transduced with anti-CD38-CAR kills AML cells and furthermore, whether the cytotoxicity is enhanced in the presence of ATRA. First of all, we confirmed that T cells bearing anti-CD38-CAR expressed GFP as well as the anti-goat mouse IgG-PerCP, whose expression is consistent with the expression of anti-CD38-CAR on the T cells. Next, we evaluated the cytotoxic effect of CD38-specific T cells against two AML cell lines (THP-1 and CMK), which highly express CD38 (〉99%). Lactate dehydrogenase (LDH) releasing assay and flow cytometry were performed for cytotoxicity. We co-incubated THP-1 or CMK cells with T cells transduced at a variety of effector (E): target (T) ratios for 3 consecutive days in vitro. Interestingly, these assays showed that T cells bearing anti-CD38-CAR were cytotoxic against THP-1 and CMK cells in dose- and time-dependent manners. However, in the cases of KG1a, U937 and HL60 cells which partially express CD38, killing effect was restricted to CD38+ cells by T cells bearing anti-CD38-CAR, and CD38− AML cells remained alive. These results suggested that augmentation of CD38 expression is essential for the sufficient cytotoxicity against AML cells not expressing CD38 by T cells bearing anti-CD38-CAR. We, then, investigated whether ATRA augmented CD38 expression and resultantly enhanced the cytotoxicity against AML cell lines. Interestingly, ATRA augmented CD38 expression in HL60 cells as well as even in KG1a and U937. However, ATRA by itself exerted no effect on cytotoxicity or proliferating activity in KG1, U937 and even in HL60 cells. These results showed that ATRA contributed to enhancement of CD38 expression, but not to cytotoxicity and proliferation on AML cell lines. Next, we attempted to examine cytotoxic effect of T cells with anti-CD38-CAR on KG1, U937 or HL60 cells following ATRA treatment. AML cell lines were co-cultured with the effector T cells transduced for 3 days in the presence of ATRA. The killing effect of T cells bearing anti-CD38-CAR against AML cell lines was limited in the absence of ATRA. Intriguingly, T cells with anti-CD38-CAR exerted enhanced cytotoxic effect on AML cells in the presence of ATRA. Next, we applied our settings above to AML cells freshly isolated from AML patients. Firstly, we confirmed that CD38 expression was enhanced by ATRA in AML cells from patients as observed in AML cell lines. Furthermore, 3-day incubation of patients' AML cells with T cells bearing anti-CD38-CAR abrogated AML cells in the presence of ATRA. These results indicated that T cells expressing anti-CD38-CAR efficiently eliminated AML cells from patients as well as AML cell line cells through the enhancement of CD38 expression by ATRA. Here, we propose that pre-treatment of ATRA in patient with AML enhances CD38 expression on the leukemic cell surface, which augments the cytotoxic effect of T cells bearing CAR to eradicate leukemic cells including LSCs. Disclosures: No relevant conflicts of interest to declare.