FL-Dependent Wild-Type FLT3 Signals Reduce the Inhibitory Effects of FLT3 Inhibitors On Wild-Type and Mutant FLT3 Co-Expressing Cells.

Abstract Abstract 2067 Poster Board II-44 Introduction: FLT3 mutation, which is found in about 30% of adult AML patients, is involved in the signaling pathway of autonomous proliferation and differentiation block in leukemia cells. Since FLT3 mutation is associated with a poor prognosis in AML patients, mutated FLT3 serves as an important molecular target in the treatment of leukemia. However, it is notable that AML cells harboring FLT3 mutation show the various degree of the allelic ratio of mutant to wild-type (wt) FLT3. To date, several FLT3 kinase inhibitors have been developed, while their inhibitory effects were mainly evaluated in mutant FLT3 alone-expressing cells. In this study, we analyzed how FL-dependent wt-FLT3 signal affects the inhibitory effect of FLT3 inhibitors on wt- and mutant FLT3-expressing cells. Methods: We first established wt-FLT3, mutant FLT3, extra cellular domain-lacking mutant FLT3 (cy-mutant FLT3), wt- and mutant FLT3 co-expressing and wt-and cy-mutant FLT3 co-expressing 32D cells. Furthermore, we also established membrane bound FL-expressing Cos7 cells. Like mutant FLT3, cy-mutant FLT3 was constitutively phosphorylated resulting in autonomous proliferation of cy-mutant FLT3-expressing 32D cells. Using these cells, we evaluated the effects of FL-dependent wt-FLT3 signal on growth inhibition, apoptosis induction and signal inhibition induced by FLT3 inhibitors. Results: The FL-stimulation reduced growth inhibitory and apoptosis induction effects by FLT3 inhibitors in wt- and mutant FLT3 co-expressing 32D cells in a dose-dependent manner, while those reducing effects were little on mutant FLT3 alone-expressing 32D cells. In consistent with the reducing effect on the growth inhibition, FL-stimulation broadly reduced the de-phosphorylation levels of FLT3, STAT5, AKT and MAPK by FLT3 inhibitors, and the strongest reduction was observed in the de-phosphorylation of MAPK. These reduced effects were more evident in the continuous FL-stimulation by the co-culture with FL-expressing Cos7 cells than the transient stimulation by the addition of soluble FL. In the transient stimulation by the soluble FL at 50 ng/ml, the growth inhibitory effects of FLT3 inhibitors were reduced to about 50% of those without the FL-stimulation. In contrast, the continuous FL-stimulation by the co-culture with FL-expressing Cos7 cells reduced the inhibitory effects to less than 40% of the co-culture with parental Cos7 cells. Of note is that the reduction level of inhibitory effects by the FLT3-selective inhibitors, such as FI-700 was greater than that by multi-kinase inhibitors, such as sunitinib. Furthermore, these FL-dependent reducing effects was also observed in wt- and cy-mutant FLT3 co-expressing cells at the same level as wt- and mutant FLT3 co-expressing cells, indicating that the FL-stimulation reduced the inhibitory effects of FLT3 inhibitors through the interaction with wt-FLT3. Conclusions: We demonstrated that FL-dependent wt-FLT3 signals reduced the inhibitory effects of FLT3 inhibitors. Since FLT3 expresses on normal hematopoietic progenitors, the lower potency against FL-dependent wt-FLT3 kinase is thought to be an advantage for avoiding the bone marrow suppression in the clinical use. However, our results suggest that the potency against both wt- and mutant FLT3 kinases is required for the sufficient efficacy of leukemia cells, which express both wt- and mutant FLT3. Furthermore, we should consider the allelic ratio of mutant to wt-FLT3 and the selectivity of FLT3 inhibitors for their clinical applications. Disclosures: Kiyoi: Kyowa Hakko Kirin Co. Ltd.: Consultancy; Novartis Pharma Co. Ltd.: Research Funding. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

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Development of Predictive Biomarker and Optimal Treatment Strategy with FLT3 Inhibitors in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-127610 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1418-1418

Author(s):

Jeong Hui Kim ◽

Yasuhiko Harada ◽

Yuichi Ishikawa ◽

Naomi Kawashima ◽

Marie Nakashima ◽

...

Keyword(s):

Research Funding ◽

Therapeutic Strategy ◽

Inhibitory Effect ◽

Genetic Alterations ◽

Molecular Characteristics ◽

Inhibitory Effects ◽

Npm1 Mutation ◽

Growth Inhibitory ◽

Flt3 Inhibitors ◽

The Impact

【Background】Recently, FLT3 inhibitors are approved for FLT3 mutated AML patients and other FLT3 inhibitors are also in clinical development. Since the target selectivity and the inhibitory activity of FLT3 inhibitors are varied, it is required to establish a therapeutic strategy in consideration of their characteristics. Most FLT3 mutated AML cells co-express wild type (Wt)-FLT3, and we previously demonstrated that FLT3 ligand (FL) stimulation attenuated the inhibitory effect of FLT3 inhibitors through the activation of Wt-FLT3 in Wt- and ITD-FLT3 co-expressing cells; however, little is known about this inhibitory mechanism in recently developed FLT3 inhibitors. In this study, we aimed to clarify the efficacy and characteristics of four FLT3 inhibitors including the impact of FL on AML cell lines and primary patients cells, and to identify biomarkers for drug selection and prediction of their efficacy. 【Methods】We examined the growth inhibitory effects of midostaurin, quizartinib, gilteritinib and FF-10101 at various concentrations of FL in ITD-FLT3 expressing 32D cells, Wt- and ITD-FLT3 co-expressing 32D cells,and FL and Wt-FLT3 co-expressing 32D cells. The inhibitory effects of these four FLT3 inhibitors were also examined in 87 primary AML cells with or without FLT3 mutationin vitro, and the correlation between their efficacy and clinical and molecular characteristics including genetic alterations and FLT3-ITD allelic ratio (ITD-AR) were investigated. Moreover, characteristics of residual AML cells after the treatment with FLT3 inhibitors were also examined in patient-derived xenografts (PDX) -AML model. 【Results】The inhibitory effect of FLT3 inhibitors was significantly impaired by FL stimulation dose dependently in all inhibitors except for midostaurin in Wt- and ITD-FLT3 co-expressing 32D cells. In FL and Wt-FLT3 co-expressing cells, GI50 value of gilteritinib was higher than that in ITD-FLT3 solo-expressing cells, and the similar tendency was observed with quizartinib. We examined the Growth inhibitory effects of these inhibitors in 33 FLT3 mutated and 54 FLT3 un-mutated primary AML cells. In primary cells, midostaurin showed lower selectivity to FLT3 mutation compared with other inhibitors. Subsequently, the correlation between the efficacy of FLT3 inhibitors and ITD-AR was examined. RNA or DNA based ITD-AR was not related to the growth inhibitory effect of FLT3 inhibitors except for gilteritinib in FLT3-ITD mutated patient cells; the GI50 value of gilteritinib in AML cells with RNA based ITD-AR-low were significantly higher than those in RNA based ITD-AR-high (P=0.036) (Figure).Moreover, FLT3-ITD cells with mutated NPM1 tend to have higher GI50values for all of FLT3 inhibitors, irrespective of ITD-AR. In AML-PDX treated with quizartinib or gilteritinib, FLT3-ITD-AR in residual AML cells was lower than that of non-treated cells, suggesting that co-expression level of Wt-FLT3 is related to the response to FLT3 inhibitors in vivo. 【Conclusions】FL affected the efficacy of FLT3 inhibitors in Wt- and ITD-FLT3 co-expressing cells, and the inhibitory effects on ITD-FLT3 and FL-Wt-FLT3 pathway were different among FLT3 inhibitors. Furthermore, NPM1 mutation and RNA based ITD-AR might be predictive biomarkers for the efficacy of FLT3 inhibitors in FLT3-ITD positive AML. The appropriate therapeutic strategy based on the characteristics of each inhibitor is necessary. Disclosures Ishikawa: Bristol-Myers Squibb: Honoraria; Abbvie GK.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Celgene Co., Ltd.: Honoraria. Goto:Celgene Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria; JCR Pharmaceuticals Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Honoraria. Ozawa:Pfizer Japan Inc.: Honoraria; Novartis: Honoraria; Kyowa-Hakko Kirin: Honoraria; Astellas Pharma Inc.: Honoraria. Kiyoi:Zenyaku Kogyo Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Pfizer Japan Inc.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding.

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FLT3 Mutant to Wild Type Allelic Ratio and Clinical Status Are Predictive of Response to FLT3 Inhibitors in AML.

Blood ◽

10.1182/blood.v114.22.1716.1716 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1716-1716

Author(s):

Mark Levis ◽

Takashi Sato ◽

Kathleen Murphy ◽

Trivikram Rajkhowa ◽

Keith Pratz

Keyword(s):

Board Member ◽

Kinase Inhibitors ◽

Clinical Status ◽

Advisory Board ◽

Wild Type ◽

Allelic Ratio ◽

Primary Blast ◽

Flt3 Inhibitors

Abstract Abstract 1716 Poster Board I-742 AML patients who harbor FLT/ITD mutations have a poor prognosis. Several small molecule kinase inhibitors with activity against FLT3 are in pre-clinical and clinical development. These compounds, derived from several different chemical classes, might be predicted to have very different effects both in vitro and in vivo on FLT3-mutant AML. We therefore compared 5 different inhibitors (Lestaurtinib, AC220, KW-2449, Sorafenib, and Sunitinib) for potency against mutant and wild type FLT3, as well as for cytotoxic effect against a series of 13 primary blast samples obtained from acute myeloid leukemia (AML) patients harboring internal tandem duplication (FLT3/ITD) mutations. Using immunoblot assays of Molm14 (FLT3/ITD, mutant) and SEMK2 (FLT3, wild type) in culture medium containing 10% bovine serum, the IC50 for inhibition of FLT3/ITD autophosphorylation for each of the drugs was as follows: Lestaurtinib 2 nM; AC220 1 nM; KW-2449 10 nM; Sorafenib 3 nM; and Sunitinib 1 nM. For wild type, these values were: Lestaurtinib 10 nM; AC220 5 nM; KW-2449 36 nM; Sorafenib 28 nM; and Sunitinib 2 nM. All 5 drugs, therefore, were more potent against the FLT3/ITD compared to wild type FLT3. Taking plasma protein binding into account, AC220 is predicted to be the most potent of these compounds in vivo (IC50 18.4 nM in plasma), while Lestaurtinib is predicted to be the least potent in vivo (IC50 700 nM in plasma). Using immunoblot assays of FLT3 in FLT3/ITD primary blast samples, we found that complete inhibition of FLT3 autophosphorylation does not always induce cell death, implying that some FLT3/ITD AML is not truly addicted to FLT3 signaling. This phenomenon was confined to patient samples obtained at diagnosis. Relapsed samples and samples with a high mutant-to-wild type allelic ratio were invariably more responsive to cytotoxicity from FLT3 inhibition compared with the samples obtained at diagnosis or those with a low mutant allelic ratio. Using an IL-3 rescue assay as well as published assays of inhibition of other kinases, we ranked the inhibitors according to their relative selectivity for FLT3. The most FLT3-selective agent was AC220, followed by sorafenib, then KW-2449, sunitinib, and finally lestaurtinib. The selectivity for FLT3 of a given inhibitor clearly influenced the cytotoxic response it induced in primary samples, in that lestaurtinib, the least selective (or most multi-targeted) was broadly cytotoxic to virtually all FLT3/ITD samples studied, while the more selective agents were more effective against samples obtained at relapse or samples with high FLT3 mutant-to-wild type ratios. FLT3 mutant AML, therefore, seems to undergo an evolution at relapse to a state of being more dependent on mutant FLT3 signaling. While multi-targeted FLT3 inhibitors may have greater efficacy in vitro, particularly in the diagnostic samples, it remains to be seen how well-tolerated such agents are clinically. Our results have important implications for the potential therapeutic use of FLT3 inhibitors. Disclosures Levis: Cephalon Inc: Clinical Advisory Board member; Ambit Biosciences Inc: Clinical Advisory Board member. Sato:Kyowa Hakko-Kirin: Employment.

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Anti–leukemia Activity of a Novel KIT Selective Inhibitor KI–328.

Blood ◽

10.1182/blood.v114.22.4146.4146 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4146-4146

Author(s):

Akane Tsujimura ◽

Yukimasa Shiotsu ◽

Hitoshi Kiyoi ◽

Yuichi Ishikawa ◽

Hiroshi Ishida ◽

...

Keyword(s):

Research Funding ◽

Kinase Inhibitors ◽

Inhibitory Effect ◽

Selective Inhibitor ◽

Inhibitory Effects ◽

Growth Inhibitory Effect ◽

Poor Prognostic Factor ◽

Kit Mutation ◽

Significant Growth ◽

Growth Inhibitory

Abstract Abstract 4146 Introduction KIT is a type III receptor tyrosine kinase together with FLT3, PDGFR and FMS. The interaction of KIT and its ligand stem cell factor (SCF) plays an important role in the cell survival, proliferation and differentiation. Activating mutations of KIT have been demonstrated in several kinds of human malignancies, such as mastocytoma, gastrointestinal stromal tumor, and acute myeloid leukemia (AML). Since KIT mutation seems a poor prognostic factor in CBF leukemia and the KIT expression is observed in most AML cells, KIT serves a molecular target for the treatment of AML. To date, several small molecules have been demonstrated to have a potency against KIT kinase, while KIT selective inhibitors are not yet developed for the clinical use. We recently developed a novel KIT selective inhibitor KI-328, and evaluated here its inhibitory effect on wild-type (Wt) and mutant KIT kinases. Methods We identified 5 types of KIT mutations (D816V, M541L, V540L, T417F/del418-419 and N822K) in AML cells, and established these mutant-KIT, as well as Wt-KIT, expressing IL-3-dependent mouse myeloid precursor 32D cells. Using these Wt- and mutant KIT expressing 32D cells, we examined the anti-leukemia activity of KI-328 in comparison with another potent KIT inhibitors. Results In Wt- and M541L-KIT expressing cells, KITs were phosphorylated by the SCF stimulation. In contrast, mutant KITs were constitutively phosphorylated in D816V-, V540L-, T417F-, and N822K-KIT expressing cells. However, the autonomous proliferation was observed only in D816V-KIT expressing cells, and the other mutant KIT expressing cells required SCF for their proliferations like Wt-KIT expressing cells. These results were confirmed by the colony formation ability in the semi-liquid media, where only D816V-KIT expressing cells could form the colony without any growth factors. The growth inhibitory effect of KI-328 was, therefore, examined in the existence of 50 ng/ml of SCF. KI-328 inhibited the growth of Wt-, M541L-, V540L-, T417F- and N822K-KIT expressing cells with the GI50 value 127 nM, 229 nM, 575 nM, 445 nM and 997 nM, respectively. The cell cycle analysis showed the KI-328 increased sub-G1 populations in these cells at each GI50 value. In consistent with the growth inhibitory effects, KI-328 potently inhibited the phosphorylations of Wt- and mutant KITs except D816V as well as their downstream molecules STAT3, AKT, MAPK at the concentration of over the GI50 value, indicating the proof of concept that KI-328 inhibits the growth of these cells by the KIT kinase inhibition. However, the significant growth inhibition was not observed in D816V-KIT expressing cells up to the 5 μM, and more than 2 μM of KI-328 were required for the de-phosphorylation of D816V-KIT. We further examined whether another potent KIT inhibitors showed the different sensitivities between D816V-KIT and Wt-KIT. Multi-kinase inhibitors such as dasatinib and sunitinib showed the same growth inhibitory effects on D816V- and Wt-KIT expressing cells: each GI50 value against D816V- and Wt-KIT was 43 nM and 72 nM, and 116 nM and 206 nM, respectively. In contrast, imatinib, which is relatively selective against KIT kinase, did not inhibit the growth of the D816V-KIT expressing cells like KI-328. Conclusions We demonstrated that KI-328 is a potent and selective KIT inhibitor. Although KI-328 did not show the significant growth inhibitory effect on the D816V-KIT expressing 32D cells up to the 5 μM, G-CSF mediating neutrophil maturation was observed when those were treated with less than 1 μM of KI-328, indicating that KI-328 has a weak potency against the D816V-KIT kinase. Therefore, the combination therapy with another potent KIT inhibitors, such as HSP90 inhibitor, might conquer the resistance against the D816V-KIT kinase. Since the kinase inhibitory profile seemed to be associated with the resistance against the D816V-KIT kinase, the structural analysis of the D816V-KIT is required for developing more potent inhibitors against all mutant KIT kinases. Disclosures: Shiotsu: Kyowa Hakko Kirin Co., Ltd.: Employment. Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Consultancy; Novartis Pharma Co. Ltd.: Research Funding. Ishida:Kyowa Hakko Kirin Co., Ltd.: Employment. Naoe:Kyowa Hakko Kirin Co., Ltd. : Research Funding; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Wyeth K.K.: Research Funding.

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The Multi-Kinase/ABL Inhibitor R763/AS703569 Induces DNA Endoreduplication and Apoptosis In Imatinib-Resistant CML Cells and Synergizes with Nilotinib, Dasatinib, and the Plk-1 Inhibitor BI 2536, In Producing Growth Inhibition.

Blood ◽

10.1182/blood.v116.21.3394.3394 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3394-3394

Author(s):

Karoline V. Gleixner ◽

Harald Herrmann ◽

Barbara Peter ◽

Katharina Blatt ◽

Karina Schuch ◽

...

Keyword(s):

Growth Inhibition ◽

Research Funding ◽

Kinase Inhibitors ◽

K562 Cells ◽

Aurora Kinase ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Imatinib Resistant ◽

Resistant Mutants ◽

Bi 2536

Abstract Abstract 3394 Resistance to imatinib is a major clinical problem and challenge in advanced chronic myeloid leukemia (CML). In most patients, drug-resistant mutants of BCR/ABL are detectable. Although most of these mutants still are responsive to second generation BCR/ABL kinase inhibitors (KI) such as nilotinib or dasatinib, drug responses are often short-lived. The BCR/ABL mutant T315I confers resistance against all available BCR/ABL KI, including nilotinib and dasatinib. More recent data suggest that several Aurora kinase (AuK) inhibitors block the kinase activity of BCR/ABL T315I. We have examined the growth-inhibitory effects of the AuK/ABL inhibitor R763/AS703569 (Merck-Serono, Darmstadt, Germany) on primary CML cells (chronic phase, n=12), the CML cell line K562, and Ba/F3 cells transfected with various imatinib-resistant mutants of BCR/ABL. As assessed by 3H-thymidine-uptake, R763/AS703569 was found to inhibit proliferation in imatinib-sensitive and imatinib-resistant primary CML cells in all donors tested, in imatinib-resistant and imatinib-responsive K562 cells, and in Ba/F3 cells harbouring various mutants of BCR/ABL (E255K, Y253F, H396P, T315I). The effects of R763/AS703569 on BCR/ABL-transformed cells were dose-dependent with IC50 values ranging between 0.001–0.1 μ M in K562 cells, <0.001-1 μ M in primary CML cells, and 0.001–0.1 μ M in BCR/ABL+ Ba/F3 cells. In all three patients in whom an imatinib-resistant BCR/ABL mutation was detected (one with V379I, one with F359V, and one with T315), R763/AS703569 was found to inhibit growth of primary CML cells at pharmacologic concentrations (IC50: 0.5, 0.005, and 0.05 μ M, respectively). As assessed by flow cytometry, the growth-inhibitory effects of R763/AS703569 were accompanied by DNA endoreduplication and consecutive apoptosis. Western blot experiments using anti-pCrkL antibody were performed and confirmed that R763/AS703569 blocks BCR/ABL activity at 1 μ M in K562 cells and BCR/ABL-transformed Ba/F3 cells. In addition, R763/AS703569 was found to block Aurora kinase A and Lyn phosphorylation in CML cells. By contrast, no effect of R763/AS703569 on phosphorylation or expression of Polo-like kinase-1 (Plk-1) was seen. In a next step, we explored the effects of drug combinations on growth of K562 cells and BCR/ABL-transformed Ba/F3 cells. In these experiments, R763/AS703569 was found to synergize with the ABL/multi-kinase inhibitors nilotinib (Novartis, Basel Switzerland) and dasatinib (Bristol-Myers Squibb, Princeton, NJ) in producing growth inhibition in CML cells (figure). Moreover, R763/AS703569 and the Plk-1 inhibitor BI 2536 (Boehringer Ingelheim, Vienna, Austria) were found to produce synergistic growth-inhibitory effects on CML cells in all samples tested. Synergistic effects of the KI applied were also seen in Ba/F3 cells exhibiting BCR/ABL T315I. In summary, our data show that the novel AuK/ABL inhibitor R763/AS703569 produces growth inhibition and apoptosis in BCR/ABL-transformed cells including those harbouring BCR/ABL T315I or other imatinib-resistant BCR/ABL mutants. Moreover, our data show that R763/AS703569 synergize with other multi-kinase/ABL inhibitors as well as with the Plk-1 inhibitor BI 2536 in producing growth inhibition in imatinib-resistant CML cells. Whether R763/AS703569, applied alone or in combination with other targeted drugs, can produces anti-leukemic effects in patients with advanced CML remains to be determined in clinical trials. Synergistic growth-inhibitory effects of R763/AS703569 and nilotinib in BCR/ABL T315I+ Ba/F3 cells (left), and R763/AS703569 and dasatinib in K562 cells (right). Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

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Mechanism of FLT3 Ligand Dependent Resistance to FLT3 Inhibitors

Blood ◽

10.1182/blood.v124.21.908.908 ◽

2014 ◽

Vol 124 (21) ◽

pp. 908-908

Author(s):

Fangli Chen ◽

Yuichi Ishikawa ◽

Hitoshi Kiyoi ◽

Tomoki Naoe

Keyword(s):

Cell Cycle ◽

Research Funding ◽

Inhibitory Effect ◽

Flt3 Ligand ◽

Inhibitory Effects ◽

Chugai Pharmaceutical ◽

Cycle Arrest ◽

Growth Inhibitory ◽

Flt3 Inhibitors

Abstract Background: FLT3mutation, which is found in about 30% of acute myeloid leukemia (AML) patients, is involved in the signaling pathway of autonomous proliferation and differentiation block in leukemia cells. Since FLT3 mutation is associated with a poor prognosis in AML patients, mutated FLT3 serves as an important molecular target in the treatment of leukemia. To date, several FLT3 inhibitors are undergoing investigation, but their clinical efficacies were lower than expected. The inhibitory effects of FLT3 inhibitors were mainly evaluated using the sole mutant FLT3-expressing cells in pre-clinical studies, while clinicallymost AML cells harboring FLT3 mutation co-express wild-type (Wt) FLT3, suggesting that FLT3 ligand (FL)-dependent Wt-FLT3 signal might cause the lower clinical efficacies of FLT3 inhibitors. Recently, it was reported that administration of FLT3 inhibitors induced increased concentration of FL in plasma. Thus, here we analyzed how FL-dependent signal affects the inhibitory effect of FLT3 inhibitors and proliferation on Wt- and ITD-FLT3-coexpressing cells. Methods: 5 kinds of FLT3-expressing 32D cells were established: Wt-FLT3, FLT3-ITD, extra cellular domain-lacking FLT3-ITD (cyFLT3-ITD), Wt- and FLT3-ITD co-expressing and Wt- and cyFLT3-ITD co-expressing 32D cells. The growth inhibitory effects of 6 FLT3 inhibitors (AC220, CEP701, FI-700, KW2449, PKC412 and Sorafenib) with and without FL stimulation were evaluated by MTT assay. Furthermore, cell cycle analysis was performed to evaluate cell proliferation, and inhibitory effects on FLT3 kinase and its downstream molecules were also evaluated by western-blot. In vivo, cells were inoculated into C3H/Hej mice intravenously to follow survival rate and NOD/SCID mice subcutaneously to compare tumor volume between sole ITD-FLT3-expressing 32D cells and Wt and ITD-FLT3-coexpressing 32D cells. Results: The FL-stimulation reduced growth inhibitory effects by FLT3 inhibitors on Wt- and ITD-FLT3-co-expressing 32D cells, while those reducing effects were little on the sole extracellular domain lacked ITD-FLT3 (cyITD-FLT3)-expressing 32D cells. Of note, FL-stimulation induced cell cycle arrest dose-dependently, resulting reduced proliferation in Wt- and ITD-FLT3-co-expressing cells. In vivo, all syngeneic C3H mice inoculated with sole cyITD-FLT3-expressing 32D cells and sole ITD-FLT3-expressing 32D cells developed leukemia within 16 days and 72 days respectively, but mice inoculated with Wt- and ITD-FLT3-co-expressing cells survived more than 100 days (P<0.0001). Furthermore, the growth of tumors driven by sole ITD- and cyITD- FLT3-expressing 32D cells was significantly faster than tumors driven by Wt- and ITD-FLT3-co-expressing cells in NOD/SCID subcutaneous model. Western blot shows AKT and ERK are activated in Wt- and ITD-FLT3-co-expressing cells by FL stimulation. STAT3 is highly phosphorylated in Wt- and ITD-FLT3-co-expressing cells and can be further activated by FL stimulation, while the phosphorylation is weak in sole ITD expressing cells. Immunopricipitation demonstrated that FLT3 ligand activated only Wt-FLT3 but not ITD-FLT3 in co-expressing cells. Furthermore, p21 (CIP1/WAF1) can be up-regulated by FL and induce cell cycle arrest in Wt- and ITD-FLT3-co-expressing cells. FL impeded the inhibitory effect of FLT3 inhibitors by persistent activation of ERK and AKT through Wt-FLT3. Also, down-regulation of p21 and Mcl-1induced by FLT3 inhibitor can be suppressed by FL. Conclusions: These results suggested that FLT3 Ligand dependent resistance to FLT3 inhibitors were associated with reduced proliferation ability caused by up-regulation of P21 and persistent ERK and AKT activation through Wt-FLT3 signal. Disclosures Kiyoi: Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Naoe:Otsuka Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding.

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Isoform Selective HDAC8 Inhibitor (OCH3) Shows Potency in Selectively Targeting Primary AML Cells

Blood ◽

10.1182/blood-2021-151599 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2944-2944

Author(s):

Amal Mechaal ◽

Amudha Ganapathy ◽

Dolores Mahmud ◽

Taha Y Taha ◽

Rajeev Ranjan ◽

...

Keyword(s):

Growth Inhibition ◽

Ex Vivo ◽

Vehicle Control ◽

Leukemia Cells ◽

Apoptotic Cells ◽

Histone H4 ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Cd34 Cells

Abstract The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific. The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP. The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%). Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Nilotinib and Dasatinib Produce Synergistic Growth-Inhibitory Effects In Imatinib-Resistant CML Cells, Including Subclones Bearing the Multi-Resistant BCR/ABL Mutant T315I

Blood ◽

10.1182/blood.v116.21.2280.2280 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2280-2280

Author(s):

Karoline V. Gleixner ◽

Harald Herrmann ◽

Irina Sadovnik ◽

Karina Schuch ◽

Winfried F Pickl ◽

...

Keyword(s):

Stem Cells ◽

Research Funding ◽

Synergistic Effects ◽

Chronic Phase ◽

Leukemic Cells ◽

Inhibitory Effects ◽

Blast Phase ◽

Drug Concentrations ◽

Growth Inhibitory ◽

Imatinib Resistant

Abstract Abstract 2280 In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (< 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined. Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

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The Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) Shows Selective In Vitro Efficacy In Monocytoid-Lineage Acute Myeloid Leukaemia (AML)

Blood ◽

10.1182/blood.v122.21.1297.1297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1297-1297 ◽

Cited By ~ 1

Author(s):

Joanna Zabkiewicz ◽

Marie Gilmour ◽

Robert K. Hills ◽

Elizabeth Bone ◽

Alan Davidson ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Bone Marrow Cells ◽

Therapeutic Window ◽

Normal Bone Marrow ◽

Inhibitory Effects ◽

Normal Bone ◽

Growth Inhibitory ◽

Deacetylase Inhibitor

Abstract Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase inhibitor (HDACi) that is cleaved to an active acid, CHR-2847, by an intracellular esterase (human carboxylesterase-1, hCE-1), found only in cells of monocytoid lineage and hepatocytes. The clinical uptake of HDAC inhibition to date has been restricted by systemic toxicities including gastrointestinal disturbance, thrombocytopenia and fatigue. Accumulation of CHR-2847 in hCE-1-expressing cells results in a 20-100-fold increase in targeted anti-proliferative potency, considerably widening the potential therapeutic window in malignancies involving cells of monocytoid lineage (AML-M4, AML-M5 and CMML) by sparing the systemic toxicological effects associated with non-selective HDAC inhibition. The in vitro efficacy of tefinostat was assessed in primary AMLs using stored mononuclear cells obtained at diagnosis from 70 AML patients. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in M4 /M5 AMLs (median IC50; 1.1µM+/-1.8) compared to non-M4/M5 FAB types (median IC50 5.1µM +/-4.7) (p=0.007). This potency and monocytoid specificity was not reproduced when using an alternative HDACi, tefinostat analogue CHR-8185 which is not cleaved by hCE-1. hCE-1 protein expression in patient samples was measured by both intracellular flow cytometry and immunoblotting, with highest levels seen in M4/M5 patients. This observation was validated by microarray analysis of hCE-1 mRNA in a further 130 AML samples with M4/M5 AMLs showing significant overexpression compared to normal bone marrow CD34+ cells (p=0.009). High levels of hCE-1 expression were found to drive a significant increase in tefinostat efficacy as measured by growth inhibition assays (p=0.001), and also strongly correlated with expression of the mature monocytoid marker CD14+. Sub-population analysis by flow cytometry revealed variable sensitivity to tefinostat within AML blasts, with CD14+ expressing cells showing maximum growth inhibition. This CD14+ response was accompanied by an induction of intracellular protein acetylation at nanomolar concentrations in tefinostat-responsive samples. Tefinostat-sensitive samples also showed strong induction of the cell cycle arrest and DNA damage sensor protein pH2AX, which is a potential biomarker of patient responsiveness. Importantly, no growth inhibitory effects were seen in normal bone marrow cells (n=5) exposed to AML-toxic doses of tefinostat while, in comparison, equivalent concentrations of the non-hCE-1-dependent analogue CHR-8185 caused considerable cytotoxicity, again emphasising the potential for expansion of the clinical therapeutic window using an hCE-1-dependent agent. In vitro synergy was demonstrated in combination experiments with tefinostat and cytarabine (median Combination Index value=0.68) which is likely to be a logical combination for future clinical evaluation. In summary, monocytoid targeting of HDACi activity was achieved using tefinostat in primary AML samples of monocytoid lineage, with minimal toxicity to normal bone marrow cells at equimolar concentrations. Given the absence of significant toxicity seen in a recently-published phase 1 study of tefinostat in patients with advanced haematological malignancies, further larger scale clinical evaluation of this compound is warranted in haematological malignancies involving cells of monocytoid lineage. Disclosures: Zabkiewicz: Chroma Therapeutics: Research Funding. Gilmour:Chroma Therapeutics: Research Funding. Hills:Chroma Therapeutics: Research Funding. Bone:Chroma Therapeutics: Employment. Davidson:Chroma Therapeutics: Employment. Burnett:Chroma Therapeutics: Research Funding. Knapper:Chroma Therapeutics: Research Funding.

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