Abstract: Abstract 3988 Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.

Abstract: Abstract 1426 Poster Board I-449 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. In order to examine mechanisms of drug resistance in LSCs and to seek strategies to overcome the resistance, we used Ph-positive acute lymphoblastic leukemia patient cells serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Recently several publications have suggested that proteasome inhibitors can induce selective cell death in LSCs. Spleen cells derived from leukemic NOG mice were treated ex vivo with imatinib and the proteasome inhibitor, bortezomib and cell viablility (PI-/Annexin-V-) was compared between treated and non-treated cells. After treatment with imatinib, significantly more residual cells were observed in the CD34+CD38- population compared to the CD34+CD38+ or CD34-CD38+ populations. With nM level of bortezomib, substantial cell death was induced in all populations with up-regulation of phospho-p53 (Ser15). Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with imatinib treatment, but not with bortezomib treatment. Regarding cell cycle states, a higher percentage of Hoechst-33342low/Pyronin-Ylow cells was observed in the CD34+CD38- population relative to the other populations, suggesting more cells in the G0 state among the CD34+CD38- population. In co-culturing with S17 stromal cells, quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib, while substantial cell death including CD34+ population was induced with nM level of bortezomib. We are also investigating more detailed biomarkers in the cell death and effects of these drugs both on the primitive leukemia cells and normal hematopoietic cells using the in vivo leukemic NOG mice systems. These results imply that resistance to imatinib in Ph-positive leukemia quiescent cells is independent of BCR-ABL phosphorylation and that treatment with bortezomib can overcome the resistance of Ph-positive LSCs. Disclosures Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe: Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

Abstract: Abstract 3277 Poster Board III-1 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells. Disclosures: Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

Abstract: Abstract 4457 Chronic myeloid leukemia (CML) is effectively treated with imatinib (IM), however, several mathematical models and ex vivo-examinations suggested that IM-therapy does not eradicate BCR-ABL-positive hematopoietic stem cells (HSC). We prospectively (0, 3, 6 and 12 months after IM-therapy) investigated 16 newly diagnosed and 22 long-term followed CML-chronic phase (CP) cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) (Figure 1) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM-therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations while gradual reduction was observed during the first 12 months in all populations. And discrepancy of minimum residual diseases (MRD) between the HSC populations and other populations was larger in patients after longer IM-therapy. In evaluating properties of CML stem cells and other markers, we observed irrelevant distribution of side population (SP) and expressions of ABC transporters (ABCB1 and ABCG2) in comparison with CD34/38 expression. We also prospectively investigated BCR-ABL transcripts in each population of 23 IM-resistant or -intolerant CML-CP cases and one newly diagnosed CML-accelerated phase (AP) case during treatment with second-generation tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months and there was no significant difference of MRD among each population in optimal responders to 2nd TKIs-therapy. In the stromal co-culturing system using primary cells and leukemic NOD/SCID/IL2rgnull (NOG) mice xenotransplanted with Ph+ leukemia cells, retention of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ population after IM-treatment were observed and cell death mechanisms after treatment with 2nd TKIs are also under investigation. These results imply that therapy with 2nd TKIs could be a promising approach for quick and efficient reduction of the CML stem cells and cure of disease. Figure 1 Figure 1. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract: Abstract 1579 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia and eradication of LSCs should be necessary for the cure. In order to examine mechanisms of drug resistance in Ph+ leukemia to imatinib (IM) due to stem cell properties and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., PNAS, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells from leukemic NOG mice were treated ex vivo with IM and growth factors, and cell viablility (PI/AnnexinV-staining) was compared between treated and non-treated cells. After treatment with IM, significantly more residual cells were observed in the CD34+/38- population compared to the CD34+/38+ or CD34-/38+ populations. Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with IM treatment. Regarding cell cycle states, a higher percentage of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) cells was observed in the CD34+/38- population relative to the other populations. Recently, aberrant activation of mTOR signaling has also been reported to be involved in LSCs. Leukemic spleen cells in longer co-culturing with stromal cells were treated with IM and mTOR inhibitor, everolimus (Eve, RAD001). While slow-cycling CD34high+ cells were insensitive to IM, combination treatment with IM and Eve induced significant cell death also in the quiescent population. In vivo-treatment with Eve decreased tumor cells in leukemic NOD mice inoculated with the leukemic NOG cells under the sub-lethally irradiated condition. Treatment with Eve also reduced long-term colony formation of Ph+ leukemia patient samples including Y253H-mutated Ph-ALL with less toxicity to normal cord blood cells. These results imply that treatment with Eve is promising for overcoming the resistance to IM due to quiescent property in Ph+ LSCs. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.

Abstract: Peripheral blood stem and progenitor cell (PBSPC) collection imposes apheresis-related risks including thrombocytopenia, hypocalcemia and thromboembolism in donors for PBSPC transplantation. Efficient PBSC collection with fewer apheresis risks and less contamination in the collected products is crucial and we have shown some distinct characteristics among apheresis devices (Transfusion, 2003, 2007 and 2014). A recently introduced apheresis device for PBSPC collections, Spectra-Optia, has not yet established supremacy in safety and efficiency over other established previous apparatuses/programs. To compare the Optia with the widely used automated program for a former apheresis machine, Spectra-Auto (Software version 6.1), we for the first time carried out a prospective randomized multicenter and crossover study for a total of 233 apheresis done from November 2013 to May 2015 in adult (≥18 years) and G-CSF-given patients/donors. The device for the first-day apheresis was randomly assigned, and a total of 152, consisting of 47 for autologous (auto-) patients and 105 for allogeneic (allo-) donors, were studied (1st-day analysis). Among them, the 81 subjects who required the second-day apheresis to collect more cells, the second-day apheresis was performed with the other machine than the one used in the first day, and apheresis collections for 2 consecutive days using both machines were compared by a paired-t test (crossover analysis). In the both devices, apheresis was performed based on the manufacturer's protocols, with 12:1 to 15:1 ratios of whole blood to acid citrate dextrose without heparin. There was no difference between the Optia (n = 74) and the Spectra-Auto (n = 78) in the pre-apheresis parameters including age, sex, circulating blood volume (CBV), and peripheral complete blood counts (CBCs). In the crossover comparison, age, sex and CBV were technically same and CBCs were similar between the two machines (n = 81 in each). The Spectra-Auto processed larger volumes compared with the Optia [P = .02 (1st-day analysis) and .08 (crossover analysis)] , although run time was similar with the two devices [P = .57 (1st-day) and .52 (crossover)]. Mild apheresis-related reactions occurred in both machines with similar frequencies of reactions, and volumes of ACD used were not different between them. Volumes of harvested products were greater in the Optia compared with the Spectra-Auto [158 vs. 95 mL, P 〈 .01 (1st-day) and 137 vs. 86 mL, P 〈 .01 (crossover)]. Yields of mononuclear cells [P = .27 (1st-day) and .22 (crossover)] and CD34+ cells [P = .10 (1st-day) and .19 (crossover)] were not different between them. However, regardless of program, pre-apheresis peripheral CD34+ cell counts strongly correlated with the number of CD34+ cells collected, and collection efficiencies of CD34+ cells were significantly higher in the Optia compared with the Spectra-Auto [82.2% vs. 66.3%, P 〈 .01 (1st-day) and 80.5% vs. 63.1%, P 〈 .01 (crossover)]. The products collected using the Optia contained more contaminating red blood cells [64 vs. 36 x 106 cells, P 〈 .01 (1st-day) and 56 vs. 33 x 106 cells, P 〈 .01 (crossover)], compared with the Spectra-Auto, without corresponding anemia. Only in crossover analysis, there was a trend that contaminated platelets were higher with the Optia compared with the Spectra-Auto (154 vs. 127 x 109 cells, P = .06). In conclusion, the Optia is tolerable, and has no significant inferiority in the yield or adverse events in apheresis collection of PBSPCs compared with Spectra-Auto. This trial was registered at http://www.umin.ac.jp/ctr/index.htm as #UMIN000012095. Disclosures Ohto: Terumo BCT: Research Funding.

Abstract: Introduction: Tyrosine kinase inhibitor (TKI) has dramatically improved the prognosis of chronic myeloid leukemia (CML) patients. Recently, many trials of TKI discontinuation revealed that approximately 40% to 60% of CML patients who received long time TKI therapy reached the treatment-free remission (TFR), thus now TFR is proposed as one of the goals in CML treatment. Achieving deep molecular response (DMR) by TKI therapy is a minimum requirement of a challenge to TKI discontinuation in CML patient, actually CML patients with molecular residual disease (MRD) showed worse consequence than undetectable MRD (IJH, 2017). On the other hand, it has been known that some patients sustain a molecular response for a long time despite BCR-ABL fusion gene positivity in their peripheral blood. We hypothesized that the residual malignant cells were eliminated by host immune systems in the patients with continuous TFR. Practically, we obtained the data that the immune responses of the patients with continuous TFR were more activated than relapsed patients in the discontinuation trial of imatinib (ASH, 2019). Here, we reported immune effects before and after TFR phase in two Japanese discontinuation trials of second-generation TKIs (JALSG N-STOP216 and D-STOP216). Methods: Japanese patients with CML-CP treated with nilotinib or dasatinib as the first line for at least three years and confirmed in DMR for at least two years were eligible. Patients who received other TKI or stem cell transplantations were excluded. Patients were re-confirmed in MR4.5 before discontinuing TKI and they were sampled peripheral blood at pre- and 1, 3 months after stopping TKIs and after retreatment (figure 1). Peripheral blood mononuclear cells (PBMCs) were subjected to staining with immune markers and analyzed by flow cytometry. Results: 51 patients treated with nilotinib and 49 patients treated with dasatinib were assessed clinical outcomes. At 12 months, 39/51 patients (76.5%) and 27/49 patients (55.1%) remained TFR in N-STOP216 and D-STOP216 respectively (EHA, 2021). For immunological analysis, 48 patients and 43 patients were analyzed by flowcytometry. We classified the patient of each trial into two groups (TFR group and Retreatment (RET) group) (figure 1). The frequency of CD4 + T cells and CD8 + T cells in CD3 + T cells was not different between both groups in each trial. The frequency of FoxP3 +CD4 + regulatory T (Treg) cells were not different between both groups in each trial. However, the kinetics of Treg cells, especially effector Treg (eTreg; FoxP3 hiCD45RA -) cells from Pre-stopping dasatinib to 1 month after stopping dasatinib, significantly increased in TFR groups but not in RET group (figure 2). This difference in kinetics of eTreg cells was not observed in nilotinib discontinuation trial (figure 2). In N-STOP216 trial, there were no differences between TFR and RET groups in the immuno-phenotype of CD8 + T cells, NK cells, or B cells, while granulocytic myeloid-derived suppressor cells (G-MDSCs) increased at 1 month after stopping nilotinib in RET group (0.27% in RET group vs. 0.08% in TFR group, P=0.011) (figure 3). On the other hand, in D-STOP216, CD8 + T cells from the patients with continuous TFR showed less exhausted phenotype (PD-1 +CD8 +T cells 37.4% in RET group vs. 49.8% in TFR group, p=0.018) and more proliferative activity (Ki67+CD8 cells 5.03% in RET group vs. 8.91% in TFR group, p=0.043) compared with RET group at 1 month after stopping dasatinib (figure 4). There were no differences in NK cells, B cells, and MDSCs between TFR and RET groups in D-STOP216. Conclusion: We found that each TKI evoked different immune responses after stopping TKI. Nilotinib has been developed as higher specific for BCR-ABL, therefore immune cells were not affected because of its narrower off-target effect. MDSCs developed by hematopoietic stem cells with BCR-ABL were decreased by the direct effect of nilotinib in the patients with continuous TFR. Antitumor immune responses might be diminished by residual MDSCs in RET group consequently it would be a molecular relapse. On the other hand, dasatinib has different immune effects probably due to its broad-spectrum kinase inhibitory effects. T cell immune responses might be exhausted by long term exposure to dasatinib in RET group. This study showed the possibility of different mechanisms of relapse caused by distinct immune effects. Figure 1 Figure 1. Disclosures Takahashi: Kyowahakko-Kirin: Research Funding; Ono: Research Funding; Asahikasei: Research Funding; Toyamakagaku: Research Funding; Eizai: Research Funding; Chugai: Research Funding; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; Meiji Seika Pharma Co, Ltd.: Honoraria; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria. Minami: Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria; Astellas: Honoraria; Ono: Research Funding; CMIC: Research Funding. Iriyama: Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Otsuka: Honoraria, Speakers Bureau. Kiyoi: Kyowa Kirin: Honoraria; Fijifilm: Honoraria; Eisai: Honoraria; Dainippon Sumitomo: Honoraria; Daiichi Sankyo: Honoraria; celgene: Honoraria; Astellas: Honoraria; Otsuka: Honoraria; Perseus Proteomics: Honoraria; Pfizer: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Zenyaku Kogyo: Honoraria. Miyazaki: Novartis: Honoraria; Abbvie: Honoraria; Kyowa-Kirin: Honoraria; Nippon-Shinyaku: Honoraria; Chugai: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Sumitomo-Dainippon: Honoraria, Research Funding; Janssen: Honoraria; Eisai: Honoraria; Daiichi-Sankyo: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Matsumura: Ono: Research Funding; Otsuka: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Shionogi: Research Funding; Taiho: Research Funding; Takeda: Research Funding; Sumitomo Dainippon: Research Funding; Nihon Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Japan Blood Products Organization: Research Funding; Mundipharma: Research Funding; AYUMI Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis: Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding; MSD: Research Funding; Mitsubishi Tanabe: Research Funding; Amgen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Kyowa Kirin: Research Funding; Eisai: Research Funding; Chugai: Research Funding; Astellas: Speakers Bureau; Asahi Kasei: Research Funding; Addvie: Research Funding.

Abstract: Chronic myeloid leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We analyzed the HSC/Progenitors profiles using fluorescence-activated cell sorting (FACS) and investigated the minimal residual disease (MRD) in HSC and myeloid progenitors from patients with CML chronic phase (CP) after IM-therapy. HSC identified as CD34+CD38–Lin–, were separated to Thy-1+ (HSC/Thy-1+) and Thy-1– (HSC/Thy-1–) cells. HSC/Thy-1+ (CD34+CD38–Thy-1+Lin–), HSC/Thy-1– (CD34+CD38–Thy-1–Lin–), common myeloid progenitors (CMP: CD34+CD38+IL-3Rα+CD45RA–Lin–), granulocyte–macrophage progenitors (GMP: CD34+CD38+IL-3Rα+CD45RA+Lin–), and megakaryocyte–erythroid progenitors (MEP: CD34+CD38+IL-3Rα–CD45RA– Lin–) were isolated by cell sorting and MRD was quantified with real-time polymerase chain reaction detecting BCR-ABL transcripts. FACS analysis revealed higher levels of the HSC/Thy-1– cells and progenitors (CD34+CD38+Lin– cells) in bone marrow from patients with CML CP than in normal bone marrow although the level of long-term HSC in CML CP was similar with normal bone marrow. After the IM-therapy the proportion of progenitor pools (CD34+Lin– cells) within Lin– were remarkably reduced, especially that of HSC/Thy-1– cells and progenitor cells. The proportion of MEP was increased and that of GMP was decreased in bone marrow from patients with CML CP as compared with their normal counterparts. BCR-ABL positive progenitors in bone marrow were eradicated within 12 month in 5 out of 9 patients. BCR-ABL positive cells, however, remained in the stem cell population. They were positive even after achieving undetectable levels of BCR-ABL transcript in total RNA isolated from the bone marrow. The ratio of BCR-ABL to BCR was significantly decreased with the continuation of imatinib, however the retention of BCR-ABL-positive cells was observed in HSC/Thy-1– or HSC/Thy-1+ populations except one out of 9 cases. These results indicated retention but significant reduction of BCR-ABL positive stem cells in CML during IM-therapy. They also implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABLpositive MRD.

Abstract: Introduction: Blinatumomab is a bispecific T-cell engager (BiTE®) targeted immuno-oncology therapy with dual specificity for cluster of differentiation (CD) 19 and CD3 that redirects the patient's CD3-positive cytotoxic T cells to lyse CD19-positive malignant cells. Global studies have evaluated blinatumomab in patients with advanced Philadelphia chromosome-negative relapsed/refractory acute lymphoblastic leukemia (Ph- R/R ALL). In the global phase 3 TOWER study, blinatumomab monotherapy vs standard-of-care chemotherapy resulted in a significantly higher rate of complete remission (CR)/CR with partial hematologic recovery of peripheral blood counts (CRh)/CR with incomplete hematologic recovery of peripheral blood counts (44% vs 25%; P 〈 0.001) and longer median overall survival (7.7 vs 4.0 months; P = 0.01) (Kantarjian H, et al. NEJM. 2017;376:836-47). There are limited data on the efficacy and safety of blinatumomab in Asian patients, whose immunologic genetic background may differ from other patient populations. Therefore, we conducted a patient-level pooled analysis of the efficacy and safety of blinatumomab in 45 Asian adult patients with Ph- R/R ALL-19 from the blinatumomab arm of TOWER (NCT02013167) and 26 from a phase 1b/2 study in Japanese adults (NCT02412306). Methods: Patients in both studies were ≥ 18 years old and had Ph- R/R ALL, 〉 5% blasts, Eastern Cooperative Oncology Group performance status 0-2, and no central nervous system pathology. Patients received a maximum of 2 cycles of induction blinatumomab for 4 weeks by continuous intravenous infusion (cycle 1/week 1: 9 μg/day; cycle 1/weeks 2-4: 28 μg/day; subsequent cycles: 28 μg/day) followed by 2 weeks of no blinatumomab (each 6-week cycle). Responders (≤ 5% bone marrow blasts within 2 induction cycles) received blinatumomab 28 μg/day up to a maximum of 5 induction/consolidation cycles. In TOWER, patients who continued morphologic remission received up to 12 months of maintenance therapy. Patients could undergo stem cell transplantation at any time following the first treatment cycle. Results: Of the 45 Asian patients enrolled (26 female; median [range] age, 43 [18-75] years; prior hematopoietic stem cell transplantation, 20 [44.4%]; ≥ 1 prior salvage therapy, 30 [66.7%] ), 44 received at least 1 cycle of blinatumomab 9-28 μg/day. Responses in the first 12 weeks of treatment (CR/CRh and minimal residual disease response) are shown in the Table. The Kaplan-Meier (KM) median overall survival time was 11.9 (95% CI: 9.9-17.1) months, and the KM median relapse-free survival time was 8.9 (95% CI: 3.8-10.7) months; median overall survival in the blinatumomab arm of TOWER was 7.7 months. Forty-one (93.2%) patients had grade ≥ 3 treatment-emergent adverse events (TEAEs), and 5 (11.4%) had fatal AEs. Grade ≥ 3 events TEAEs of interest included neurologic events (4.5%), cytokine release syndrome (2.3%), cytopenias (6.8%), and infections (20.5%). Conclusions: The safety and efficacy of blinatumomab in Asian patients were comparable with previous global studies with similar disease response rates and a favorable safety profile with no new safety signals. Disclosures Kobayashi: Astellas Amgen BioPharma: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; SymBio: Consultancy. Iida:Chugai Pharmaceutical Co., Ltd.: Research Funding. Minami:Astellas: Research Funding; Bayer: Honoraria, Other: Clinical trial, Research Funding; Taiho: Honoraria, Other: Clinical trial, Research Funding; Taisho-Toyama: Research Funding; Takeda: Honoraria, Research Funding; CSL Behring: Research Funding; Genomic Health: Honoraria; Daiichi Sankyo: Other: Clinical trial, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Eizai: Honoraria, Research Funding; Janssen: Honoraria; Kowa: Honoraria; Kyowa-Kirin: Honoraria, Research Funding; Nihon Shinyaku: Research Funding; Eli Lilly: Honoraria, Research Funding; Merck Serono: Honoraria, Research Funding; MSD: Honoraria, Other: Clinical trial, Research Funding; Nippon Chemiphar: Honoraria, Research Funding; Ono Yakuhin: Honoraria, Other: Clinical trial, Research Funding; BMS: Honoraria, Other: Clinical trial, Research Funding; Celgene: Honoraria; AstraZeneca: Other: Clinical trial; Boehringer: Honoraria, Research Funding; Otsuka: Honoraria; Pfizer: Honoraria, Other: Clinical trial, Research Funding; Sanofi: Honoraria, Research Funding; Shire Japan: Honoraria; Abbvie: Honoraria; Nihon Medi-Physics: Honoraria; Asahi-Kaseo Pharma: Research Funding; Amgen Inc: Other: Clinical trial; Nihon Kayaku: Research Funding; Shionogi: Research Funding; Novartis: Honoraria, Other: Clinical trial; Chugai: Honoraria, Other: Clinical trial, Research Funding; Yakult: Research Funding; Teijin Pharma: Research Funding. Maeda:Mundipharma Co Ltd.: Honoraria; Kyowa Kirin Co. Ltd.: Honoraria; Astellas Pharma Inc.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Yoon:Novartis: Consultancy, Honoraria; Yuhan Pharma: Research Funding; MSD: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Janssen: Consultancy; Amgen: Consultancy, Honoraria. Tran:Amgen: Employment, Equity Ownership. Morris:Amgen: Employment, Equity Ownership. Franklin:Amgen: Employment, Equity Ownership. Chong:Amgen Asia Holding Limited: Employment, Equity Ownership. Kiyoi:Astellas Pharma Inc.: Honoraria, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; FUJIFILM Corporation: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Perseus Proteomics Inc.: Research Funding.

Abstract: Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell (LSC) populations in several experimental systems. Cumulative evidence suggests that dormant self-renewing LSC contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. PF-04449913 (PF) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF has shown promising Results regarding safety, tolerability, and early signs of efficacy in a phase 1 dose-escalation study of hematologic malignancies including AML (Jamieson C, et al. ASH, 2011). On the basis of these encouraging Results, phase 1b and phase 2 studies of PF in combination with chemotherapies have been planned in patients with AML. However, the detailed mechanisms and biomarkers remain to be elucidated in AML therapy with Hh pathway inhibitors. Research Aims and Methods We used AML cell lines and patient-derived primary AML cells in order to evaluate the efficacy and elucidate detailed mechanisms and biomarkers in the Hh antagonist, PF treatment. Using the co-culturing system with HS-5 stromal cells, the colony assay system, and the immunodeficient NOD/SCID/IL2rgnull (NOG) mouse model serially xenotransplanted with primary AML cells, we examined the effects of PF on LSC population and AML propagation. Results Using FACS sorting and RQ-PCR assays of AML patient-derived primary cells, the Hh signaling pathway was activated more in CD34-positive cells than CD34-negative cells. Ex vivo-treatment with PF inhibited proliferation and induced minimal cell death in leukemia cell lines and primary AML cells. However, in vivo-treatment with PF attenuated leukemia-initiation potential in AML cells through the serial transplantation system, while limiting reduction of tumor burden in the primary leukemia system. Also in the colony-assay system using primary AML cells, treatment with PF reduced serially colony formation. In MOLM-14 cells, treatment with PF down-regulated mRNA encoding downstream effector GLIs and GLI-targeting molecules in the canonical Hh pathway using RQ-PCR assays, and decreased nuclear expression of GLI-2 using immunofluorescence assays. In addition, treatment with PF remarkably decreased the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population and increased cycling cell population. In the in vivo-NOG mouse system, comprehensive Gene Set Enrichment Analysis (GSEA) revealed that PF treatment modulated cell cycle regulation and self-renewal signaling in primary AML cells. Moreover, combined treatment with PF abrogated resistance to Ara-C in AML cell lines co-cultured with HS-5 stromal cells and sensitized primary AML cells to Ara-C in the colony-assay system. We are also investigating toxicity for normal cord blood cells with PF treatment. Conclusions Our findings imply that selective Hh inhibitor, PF treatment can attenuate the leukemia-initiation potential in AML cells by modulation of cell cycle regulation and self-renewal signaling, and can also improve AML therapy through sensitizing dormant LSC to chemotherapy and overcoming the resistance in the bone marrow microenvironment. Disclosures: Kiyoi: Kyowa Hakko Kirin Co. Ltd.: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding.