scholarly journals The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4044-4044
Author(s):  
Blake S Moses ◽  
Jennifer Fox ◽  
Xiaochun Chen ◽  
Samantha McCullough ◽  
Sang Ngoc Tran ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Leukemia Cell ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

scholarly journals Development of Astatine-211 (211At)-Based Anti-CD123 Radioimmunotherapy for Acute Leukemias and Other CD123+ Hematologic Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3341-3341
Author(s):  
George S. Laszlo ◽  
Johnnie J. Orozco ◽  
Allie R. Kehret ◽  
Margaret C. Lunn ◽  
Donald K. Hamlin ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Efficacy Studies

Abstract Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P<0.0001 for Ctrl vs. 10 µCi, P<0.004 for 10 µCi vs. 40 µCi), demonstrating potent in vivo anti-leukemia efficacy of a single dose of 211At-CD123 RIT. Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

scholarly journals Concomitant Targeting of FLT3 and BTK with CG'806 Overcomes FLT3-Inhibitor Resistance through Inhibition of Autophagy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2635-2635
Author(s):  
Weiguo Zhang ◽  
Guopan Yu ◽  
Hongying Zhang ◽  
Charlie Ly ◽  
Bin Yuan ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Board Of Directors ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Induced Apoptosis ◽  
Inhibitor Resistance

Abstract Fms-like tyrosine kinase 3 (FLT3)-targeted therapy represents an important paradigm in the management of patients with highly aggressive FLT3 mutated acute myeloid leukemia (AML). However, clinical efficacy is usually transient and followed by emergence of resistance to FLT3-inhibitors (Borthakur et al., 2011; Cortes et al., 2013; Zhang et al., 2008). Such resistance often results from acquired mutations of TKD, which are frequently identified in D835, Y842 and F691 residues (Smith et al., 2015; Smith et al., 2012; Zhang et al., 2014). It was reported that the FLT3-ITD-targeting drug sorafenib can induce autophagy in human myeloid dendritic cells (Lin et al., 2013). Induction of autophagy has also been reported to play a crucial role in resistance to BCR-ABL targeted imatinib therapy in CML (Hekmatshoar et al., 2018). Additionally, inhibition of autophagy can re-sensitize cancer cells to apoptosis induction (Fitzwalter et al., 2018; Piya et al., 2017), suggesting that inhibition of autophagy may represent a novel therapeutic strategy for overcoming resistance to FLT3-targeted therapy. In the present study, we assessed autophagy levels in leukemia cell lines bearing different FLT3 mutations and in AML patient samples obtained from sorafenib-resistant patients. All tested resistant cell lines bearing TKD or ITD+TKD mutations showed increased basal autophagy levels. Resistant AML patient samples also demonstrated greater autophagy compared to matched pre-treatment samples in FLT3-mutated, but not in FLT3-wild type samples. Upregulation of autophagy was also observed in the bone marrow (BM)-mimetic microenvironment (i.e., hypoxia and the presence of mesenchymal stem cells (MSCs) in vitro. Inhibition of autophagy with chloroquine (CQ) potentiated quizartinib-induced apoptosis and partially abrogated MSC-mediated protection in FLT3-ITD- and/or D835-mutated AML cells by suppressing c-Myc, mTOR/S6K signaling and activating transcription factor 4 (ATF4). We also observed upregulation of BTK activation accompanied by increased autophagy levels in hypoxic/MSC co-culture with leukemic cells and in resistant primary patient samples. Co-targeting BTK and FLT3 with ibrutinib (or BTK siRNA) and quizartinib enhanced leukemic cell killing and abrogates MSC-mediated protection of FLT3 mutated leukemia cells. We further investigated a novel, highly potent small molecule pan-FLT3/pan-BTK kinase inhibitor CG-806 (IC50s 0.8 and 5.0 nM against FLT3-ITD and BTK, respectively) (Aptose, San Diego, CA). CG'806 abolished MSC/hypoxia-mediated protection of AML cells and induced apoptosis in FLT3-mutated cells in vitro. Of note, CG'806, but not quizartinib, exerted profound pro-apoptotic effects in primary AML patient cells harboring ITD+D835 mutations ex vivo. Further evaluation in a PDX leukemia model inoculated with the ITD+D835 mutated primary AML cells showed that CG'806 significantly reduced leukemia cell burden and benefited for mouse survival. Taken together, autophagy is associated with AML resistance to FLT3-targeted therapy, which can be overcome by the pan-FLT3/pan-BTK kinase inhibitor CG-806 through concomitant blockade of FLT3 and BTK. Co-targeting FLT3 and BTK might provide a strategy for preventing/overcoming FLT3 inhibitor resistance in AML patients with FLT3 mutations. Phase I trials of CG'806 are in preparation. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Rice:Aptose Biosciences, Inc: Equity Ownership. Andreeff:Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy.

scholarly journals Selective Targeting of Histone Deacetylase 11 Disables Metabolism of Myeloproliferative Neoplasms

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 474-474
Author(s):  
Vasundhara Sharma ◽  
Lanzhu Yue ◽  
Nathan P. Horvat ◽  
Agni Christodoulidou ◽  
Afua Adutwumwa Akuffo ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Cell Types ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Leukemia Cell Lines

Introduction: Acetylated histone and non-histone proteins are pharmacologic targets for both solid and hematological cancers including myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies driven by aberrant JAK2/STAT signaling. MPNs are characterized by epigenetic alterations, including aberrant acetylation, which makes this disease particularly interesting for targeting with HDAC inhibitors. Four classes of histone deacetylases (Class I-IV HDACs) regulate gene transcription and modulate cellular processes that drive the initiation and progression of cancer. Pan-HDAC and class I-selective HDAC inhibitors have gained traction in clinical settings, yet we reasoned that specific targeting of the 18 distinct HDAC proteins may establish roles for select HDACs as therapeutic vulnerabilities in MPNs. Methods: To explore the roles of individual HDACs in MPN, we first conducted an inhibitor screen of compounds having distinct HDAC selectivity based on electrophoretic mobility shift assays with full-length human HDAC proteins expressed in baculovirus and unique peptide substrates. Ultra-specific HDAC6 compounds were initially targeted for analysis based on its previously defined role in HSP90-mediated JAK2 stabilization and translation. Survival of MPN cell line models, MPN patient samples, leukemia cell lines, and MPN disease progression in mice transplanted with Hdac6-/-, and Hdac11-/- hematopoietic stem cells (HSCs) transduced with the MPLW515L oncogene, as well as Tg-Hdac11-eGfp mice were used to show the role of HDAC6 and HDAC11 in oncogene-driven and homeostatic hematopoiesis. As further proof of specificity, HDAC6 and HDAC11 were genetically ablated in MPN model cell lines using either RNA interference or inducible shRNA. For HDAC11 substrate identification, a combination of RNA-seq, acetylated proteome (SILAC), global metabolomics (LC-MS), Seahorse metabolic assays (Agilent Technologies), enzymatic assays, and acetylation-specific immunoblotting and mutation profiling were performed (Fig. 1). Results: Despite the established interplay between HDAC6, HSP90 and JAK2, neither a highly selective HDAC6 inhibitor, HDAC6 silencing, nor the Hdac6 deficiency suppressed MPN pathogenesis, although there were clear effects on the acetylation of α-tubulin, a well characterized HDAC6-selective substrate. Intriguingly, both inhibition of HDAC11 activity with highly-specific HDAC11 inhibitors and silencing HDAC11 using an inducible validated shRNA, identified HDAC11 as a therapeutic vulnerability for multiple human MPN cell lines. The Tg-Hdac11-eGFP reporter mice showed that HDAC11 is expressed in several hematopoietic cell types, including myeloid cells, erythroblasts, and megakaryocytes. Thus, Hdac11-/- and Hdac11+/+MPLWT bone marrow were examined for steady-state hematopoiesis and transplantation chimerism. These studies demonstrated that HDAC11 does not contribute to homeostatic or transplantated bone marrow reconstitution. However, in the oncogenic MPL model, recipient mice transplanted withoncogenic MPLW515L-expressing Hdac11-deficient HSCs displayed markedly impaired cytokine-independent colony-formation, had less fibrosis, and displayed improved survival in primary and secondary MPN hematopoietic stem cell transplantation; thus HDAC11 contributes to MPN pathogenesis (Fig. 1). Studies in additional leukemia cell lines, including THP-1, HL-60, and mantle lymphoma cell lines, but not in Ramos or K562 cells, established that HDAC11 contributes to oncogene-driven events in other cell types. Mechanistically, RNA-seq, SILAC proteomics, and metabolic profiling revealed that HDAC11 controls aerobic glycolysis by deacetylating Lys343 of the glycolytic enzyme enolase-1 (ENO1), functionally inactivating ENO1. Finally, the effects of targeting HDAC11 on metabolism were augmented by blocking compensatory pathways of oxidative phosphorylation that are induced via JAK2V617Fand MPLW515L oncogenic signaling. Conclusions: Our comprehensive screens of HDAC inhibitors, coupled with our biological, in vivo and molecular studies, indicate that HDAC11 is an attractive and potent target for disabling MPN metabolism and pathogenesis. These finding support the rationale for further development of clinical HDAC11 inhibitors for the treatment of metabolically-active cancers such as MPNs. Disclosures Pinilla Ibarz: Teva: Consultancy; TG Therapeutics: Consultancy; Sanofi: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Reuther:Incyte Corporation: Research Funding. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Lilly: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria. Verma:BMS: Research Funding; Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Epling-Burnette:Incyte Corporation: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Forma Therapeutics: Research Funding.

scholarly journals Development of Cirmtuzumab Antibody-Drug Conjugates (ADCs) Targeting Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1862-1862 ◽  
Author(s):  
Yousaf A. Mian ◽  
George F. Widhopf II ◽  
Thanh-Trang Vo ◽  
Katti Jessen ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Half Life ◽  
Research Funding ◽  
Leukemia Cell ◽  
Volume Distribution ◽  
Advisory Committees ◽  
High Affinity

Abstract ROR1 is an onco-embryonic surface antigen expressed on chronic lymphocytic leukemia (CLL) and a variety of other cancers, but not on most normal adult tissues. We generated a humanized IgG1 monoclonal antibody (mAb) cirmtuzumab (formerly UC-961) that binds with high affinity to a specific extracellular epitope of human ROR1 and that can block Wnt5a-induced ROR1 signaling (Yu, J et al, J Clin Invest126:585, 2016; Yu, J et al, Leukemia31:1333, 2017). Preclinical studies found that cirmtuzumab did not react with normal post-partem cells and had a pharmacokinetic (PK) volume distribution in primates consistent with a lack of off-target binding to normal tissues. We evaluated cirmtuzumab in a phase I clinical trial involving patients with relapsed-refractory CLL (Choi MY, et al, Cell Stem Cell22:951, 2018); the drug was well-tolerated at doses ≤20 mg/kg (highest dose tested) without dose-limiting toxicity. PK studies showed cirmtuzumab had a half-life of 32.4 days with no evidence for development of neutralizing antibodies or off-target sequestration of infused antibody. Furthermore, cirmtuzumab effected partial down-modulation of leukemia-cell ROR1 in patients treated with doses ≥2 mg/kg. In vitro confocal microscopy studies showed that this down-modulation was caused by internalization of cirmtuzumab-ROR1 complexes into lysosomal compartments and concomitant steady-state re-expression of nascent surface ROR1. Because of its high specificity, in vivo stability, long serum half-life, and potential capacity to concentrate conjugated drugs into lysosomal compartments, cirmtuzumab appeared ideally suited to serve as the targeting moiety in anti-ROR1 ADCs. We therefore examined cirmtuzumab-based ADCs in collaboration with VelosBio Inc., evaluating multiple linker/payload chemistries, both as single agents and in combinations. We selected for further testing cirmtuzumab-ADC-7, a cirmtuzumab-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding specificity of cirmtuzumab and allows for ROR1-targeted intracellular release of MMAE. We found cirmtuzumab-ADC-7 was selectively cytotoxic for ROR1+ CLL and mantle-cell lymphoma (MCL) cell lines at nM concentrations in vitro. Moreover, cirmtuzumab-ADC-7 caused dramatic and sustained in vivo clearance of adoptively-transferred ROR1+ leukemia cells generated from ROR1xTCL1 transgenic mice (Widhopf G, et al, PNAS111:793, 2014), ROR1+ MCL-xenografts, or ROR1+ cancer patient-derived xenografts (PDX). Further, treatment caused dose-dependent and statistically significant decreases in total cancer burden with complete regressions of tumor in multiple animals; no effect on tumor-clearance was observed in mice treated with a control MMAE-ADC of irrelevant specificity. Recently we identified that miR-15a/16-1, which commonly are deleted/downregulated in CLL, target both BCL2 and ROR1, thereby accounting in part for the direct relationship we observed between the levels of BCL2 and levels of surface ROR1 expressed by CLL of different patients (Rassenti, LZ, et al,PNAS114:10731, 2017). Because high level expression of BCL2/ROR1 may mitigate the cytotoxic activity of the BCL2-antagonist venetoclax, but potentially enhance the cytotoxicity of cirmtuzumab-ADC-7, we treated ROR1+ leukemia/lymphoma cell lines with venetoclax and/or cirmtuzumab-ADC-7. Chou-Talalay combination indices were <0.5 in all ROR1+ cell lines tested, indicating strong antitumor synergy with these two agents. Collectively these data support the rationale for clinical development of a cirmtuzumab-based ADC for treatment of patients with ROR1+ malignancies. Disclosures Vo: VelosBio: Employment. Jessen:VelosBio: Employment. Kipps:Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Verastem: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Bromodomain and Extra Terminal Domain (BET) Inhibitors Sensitize Chronic Myelomonocytic Leukemia (CMML) to PIM Inhibition Via Downregulation of Mir-33a

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4220-4220
Author(s):  
Christopher Letson ◽  
Alexis Vedder ◽  
Ariel Quintana ◽  
Phillip Liu ◽  
Brett Reid ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Bet Inhibitors ◽  
Dose Dependent

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

scholarly journals Loss of KDM6A Confers Drug Resistance in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3935-3935
Author(s):  
Sophie M. Stief ◽  
Anna-Li Hanneforth ◽  
Raphael Mattes ◽  
Sabrina Weser ◽  
Binje Vick ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Rna Seq ◽  
Advisory Committees ◽  
Leukemia Cell Lines

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML. In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells. To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1. To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity. In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. Disclosures Hiddemann: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

A Phase II Pilot Study of Kinase Inhibition in Relapsed/Refractory Acute Leukemias: Using an in Vitro Kinase Inhibitor Panel to Select Individualized, Targeted Therapies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4051-4051
Author(s):  
Stephen E Spurgeon ◽  
Rachel Cook ◽  
Elie Traer ◽  
Uma Borate ◽  
Richard T. Maziarz ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Clinical Activity ◽  
Advisory Committees ◽  
In Vitro Sensitivity ◽  
Equity Ownership ◽  
Correlative Studies

Abstract Despite improved understanding of molecular lesions in relapsed/refractory acute leukemia, outcomes remain dismal. Aberrantly activated tyrosine kinase signaling pathways play a critical role in the pathogenesis of acute leukemia. Identification of drugs that target the drivers of disease has the potential to improve treatment. We have developed an FDA regulated (IDE# G110198) in vitro kinase inhibitor assay that can identify potential therapeutic targets in primary acute leukemia samples and provide individualized treatment options in a clinically relevant time frame. We designed a Phase 2 clinical trial (NCT01620216) to validate the role of this pre-clinical kinase inhibitor screen for selecting effective individualized therapies. After obtaining informed consent, peripheral blood or bone marrow is isolated using a ficoll-density gradient and plated with dose-escalating concentration gradients of five kinase inhibitors (nilotinib, dasatinib, sorafenib, sunitinib, and ponatinib). Cell line controls are used for each drug. Samples are deemed sensitive based on IC50 and comparison to the median of all samples tested in our laboratory (over 1000 primary samples to date). Patients who satisfy eligibility are treated with FDA approved dosing using the selected inhibitor. Inclusion is limited to patients age ≥ 21 with relapsed/refractory acute leukemia (AML and ALL) and patients age ≥ 65 with a history of myelodysplasia who have developed AML, have failed hypomethylating agents, and are not candidates for standard induction. Only patients with samples that demonstrate in vitro sensitivity to 1 of the trial drugs are eligible. Additional eligibility includes: ECOG ≤ 2, adequate organ function, and no active GVHD. Cycles are 28-days. Marrow biopsy for response assessment and correlative studies is obtained on days 15, 28, and day 1 of subsequent cycles. The primary objective is to determine the clinical activity, defined as > 25% decrease in bone marrow blast counts. Secondary objectives include overall response (defined by the International Working Group), overall survival, and progression free survival. Correlative studies include high-throughput sequencing, expression profiling, correlation of target inhibition with clinical response, and PK analysis. The treatment approach is deemed worthy of ongoing study if clinical activity is observed in at least 4 of 24 patients. 43 patients have been screened with 12 demonstrating in vitro sensitivity. Nine have been enrolled on treatment. Median age is 64 (31-71) years with 5 males and 4 females. Two patients had ALL. The remainder had AML. Drugs utilized to date include dasatinib (n=3) and sorafenib (n=6). Of the three patients who exhibited in vitro sensitivity but were not enrolled, sensitivity to sorafenib, sunitinib, and dasatinib was seen. Clinical activity was seen in 4 subjects with AML -all treated with sorafenib- with median change in marrow blast in this group of 69% (44-95%). Median time on treatment for responding patients was 39 days (18-110 days). One subject with refractory AML who had failed allogeneic transplant had a 95% decrease in marrow blasts with prolonged disease control (Figure 1). Two patients were taken off study due to drug-related AEs (pancreatitis and diarrhea). No unanticipated AEs were seen. Our in vitro kinase inhibitor assay identified potential therapies for the treatment of ALL and AML. Although the numbers are too small to draw any specific conclusions regarding response, the 4 responders were FLT3-ITD+ AML, a known target of sorafenib, and appear to have proliferative disease with higher baseline blast counts. The relatively small panel of available treatment drugs with overlapping target profiles may have contributed to the preferential treatment of patients with FLT3-ITD+ AML and to the significant screen fail rate. Our results using a larger panel of targeted agents have shown a high level of sensitivity to at least one drug. Accordingly, we are expanding the platform of clinically available targeted agents. The study continues to enroll patients with the goals of defining additional molecular abnormalities and optimizing assay characteristics, while prospectively testing the therapeutic potential of additional drugs. Correlative studies are ongoing. We have initiated another study using this assay to help select targeted therapies to be added to AML induction (NCT02779283). Figure Figure. Table Table. Disclosures Spurgeon: Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Maziarz:Incyte: Membership on an entity's Board of Directors or advisory committees; Athersys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Tyner:Janssen Research & Development: Research Funding; Inctye: Research Funding; Genentech: Research Funding; Constellation Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy; Agios Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Takeda Pharmaceuticals: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Hydroxyurea Induces Growth Inhibition in BCR-ABL1 T315I+ Clones and Synergizes with Ponatinib in Killing TKI-Resistant CML Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5425-5425
Author(s):  
Mathias A Schneeweiss ◽  
Konstantin Byrgazov ◽  
Chantal B Lucini ◽  
Susanne Herndlhofer ◽  
Wolfgang R. Sperr ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
In Vitro Studies ◽  
Thymidine Uptake ◽  
Single Mutation ◽  
Mrna Levels ◽  
Advisory Committees

Abstract In chronic myeloid leukemia (CML), the occurrence of BCR-ABL1 T315I is associated with resistance against first- and second-generation BCR-ABL1 tyrosine kinase inhibitors (TKI). Ponatinib is a third generation TKI that exerts strong anti-neoplastic effects in advanced CML and is capable of suppressing the kinase activity of BCR-ABL1 T315I. However, therapy with ponatinib is associated with potentially severe side effects. In addition, resistance against ponatinib may develop in sub-clones carrying multiple (compound) mutations in BCR-ABL1. In addition, BCR-ABL1-independent oncogenic pathways contribute to drug resistance. For these patients, alternative therapies such as stem cell transplantation (SCT) or various drug combinations are often considered.Hydroxyurea (HU) is used for initial or palliative cytoreduction in CML. However, the effects of HU on TKI-resistant mutant sub-clones have not been examined so far. The aims of this study were to explore the effects of HU on CML clones carrying BCR-ABL1 T315I as individual mutation or in compound-context, and to investigate anti-leukemic effects of the drug combination ponatinib+HU. In in vitro studies, primary patient-derived cells, human CML cell lines (K562, KU812, KCL-22), and Ba/F3 cells expressing wild type (wt) BCR-ABL1, BCR-ABL1 T315I, or BCR-ABL1 compound mutants involving T315 were examined. Cell proliferation was quantified by measuring 3H-thymidine uptake. Drug effects on competitive clonal growth were analyzed by mixing two Ba/F3 clones, one expressing BCR-ABL1 T315I with GFP and one BCR-ABL1 T315I/E255V labeled by tdTomato, at a 1:1 ratio. Then, cells were exposed to HU, pontinib, or HU+ponatinib for 72 hours, and the percentage of viable cells in each clone was analyzed by flow cytometry. The in vivo response of primary CML cells carrying BCR-ABL1 T315I to HU was examined in 4 TKI-resistant CML patients who were treated with HU (1-3 g/day) for up to 18 months. In these patients, we measured white blood counts (WBC), differential counts, and BCR-ABL1 transcript levels in peripheral blood (PB) by qPCR. The percentage of BCR-ABL1 T315I compared to total BCR-ABL1 was determined by ligation-dependent PCR. In all 4 patients treated with HU, WBC and total BCR-ABL1 mRNA levels remained stable for 3-12 months. Surprisingly, in 3 of 4 patients, the leukemic sub-clone expressing BCR-ABL1 T315Iwas no longer detectable after HU-treatment. After 3 months, 2/4 patients received allogenic SCT. In the other 2 patients, the disease remained stable for 6 and 12 months, respectively. In our in vitro studies, HU was found to inhibit the growth of all BCR-ABL1+ cell lines, including K562 (IC50: 1120±89 µM), KU812 (IC50: 216±32 µM), and KCL-22 (IC50: 196±23 µM) as well as Ba/F3 cells harboring BCR-ABL1 T315I as single mutation (IC50: 74±25 µM) or as compound together with E255V (IC50: 86±2 µM), F311L (IC50: 76±20 µM), F359V (IC50: 69±10 µM), or G250E (IC50: 89±4 µM). Interestingly, Ba/F3 cells exhibiting BCR-ABL1 T315I alone or in compound configuration were more sensitive to HU compared to Ba/F3 cells expressing wt BCR-ABL1 (IC50: 236±49 µM). As expected, HU was also found to inhibit growth of primary CML cells. In subsequent experiments, HU and ponatinib were found to synergize with each other in inhibiting growth of K562, KU812, and KCL-22 cells as well as Ba/F3 cells carrying BCR-ABL1 T315I (Figure) or BCR-ABL1-T315I/F359V. In cell mix experiments, ponatinib exerted strong growth-inhibitory effects on Ba/F3-T315I cells but not on Ba/F3-T315I/E255V cells, whereas HU was found to produce stronger effects on Ba/F3-T315I/E255V cells, and only the combination of both drugs resulted in complete suppression of both cell lines. In conclusion, HU exerts strong sub-clone-specific anti-neoplastic effects in TKI-resistant CML cells, both in patients with BCR-ABL1 T315I+CML and in various cell line models, including sub-clones harboring BCR-ABL1 T315I as single mutation or in compound configuration. In addition, we show that HU and ponatinib produce strong synergistic anti-neoplastic effects on TKI resistant CML cells, including sub-clones carrying T315I. These observations may have clinical implications and may pave the way for more effective sub-clone-eradicating but also palliative or bridging-to-SCT concepts in advanced CML. Clinical studies are now warranted to define the exact value of the drug combination ponatinib+HU in TKI-resistant CML. Figure 1 Figure 1. Disclosures Sperr: Novartis: Honoraria; Amgen: Honoraria, Research Funding. Lion:Ariad: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Hoermann:Ariad: Honoraria; Novartis: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Valent:Amgen: Honoraria; Novartis: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

TGR-1202 Suppresses AML and ALL Cells Via Selective Inhibition of PI3Kδ Kinase.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2610-2610 ◽  
Author(s):  
Swaroop Vakkalanka ◽  
Srikant Viswanadha ◽  
Robert Niecestro ◽  
Peter Sportelli ◽  
Michael Savona
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Whole Blood ◽  
Leukemia Cell ◽  
Pharmacokinetic Studies ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Leukemia Cell Lines ◽  
Pharmacokinetic Properties ◽  
Equity Ownership

Abstract Abstract 2610 Background: Acute leukemia, characterized by the presence clonal hematopoietic cells in peripheral blood and bone marrow, comprises approximately 40% of newly diagnosed leukemias. First line treatment for acute leukemias with multi-agent cytotoxic chemotherapy is usually associated with significant toxicity. Advances in therapy have been slow, and nearly all effective therapies lead to prolonged marrow suppression and toxicities associated with subsequent cytopenias. Herein, we describe the biological and pharmacokinetic properties of TGR-1202, a novel small molecule PI3Kδ inhibitor with scope to be developed as a safe and effective therapy for acute myeloid (AML) and lymphoblastic (ALL) leukemia. Material & Methods: Activity of TGR-1202 against individual isoforms of the PI3K enzyme was determined via enzyme, cellular, and whole blood based assays. Potency of the compound was confirmed via leukemic cell viability and Annexin V/PI staining besides testing for inhibition of pAkt, a downstream kinase regulating cell survival and growth. These assays were conducted with cell lines (CCRF-CEM, HL-60, and MOLT-4) and patient derived cells. Anti-tumor efficacy of the compound was studied in vivo with the subcutaneous MOLT-4 xenograft model. Lastly, ADME and pharmacokinetic properties of the molecule were determined. Results: TGR-1202 demonstrated significant potency against PI3Kδ (22.2 nM) with several fold selectivity over the α (>10000), β (>50), and γ (>48) isoforms. Additionally, the compound inhibited B-cell proliferation (24.3 nM) and FcεR1 induced CD63 expression in human whole blood basophils (68.2 nM) indicating specificity towards the delta isoform. Viability testing demonstrated that the compound caused a dose-dependent inhibition in growth of immortalized as well as patient-derived AML and ALL cells. Reduction in viability was accompanied by a reduction in pAKT (>50% @ 0.3–1 μM) along with a significant induction in apoptosis in both cell lines (CCRF-CEM, HL-60, and MOLT-4) and patient samples. In tumor xenografts, oral administration of 150 mg/kg RP5264 salt over a 25-day period resulted in significant inhibition (>50%) of MOLT-4 tumor growth in mice. Pharmacokinetic studies across species indicated good oral absorption (>40% bioavailability for mice, rat, and dog) with favorable plasma concentrations (3–10 μM @ 20 mg/kg for mice, rat, and dog) relevant for efficacy. In addition, early toxicological evaluation of the molecule indicated a MTD > 500 mg/kg over a 14-day treatment period in Balb/c mice. Conclusions: TGR-1202, primarily, through its activity at the δ isoform of PI3K, has activity in both myeloid and lymphoid acute leukemia cell lines and primary patient tumors. Further evaluation of this molecule in the treatment of AML and ALL is justified, and current testing of TGR-1202 in various leukemia cell lines and within a variety of primary leukemias is ongoing. Disclosures: Vakkalanka: Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Niecestro:TG Therapeutics, Inc.: Consultancy, Equity Ownership. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership.

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