scholarly journals Identification of Specific Hnrnps As Novel Therapeutic Targets and Responsive Indicators of KPT330 (selinexor) in Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1657-1657 ◽  
Author(s):  
Adam Cloe ◽  
Li Chen ◽  
Yuan Li ◽  
Hongtao Liu ◽  
Jason X. Cheng
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Nuclear Accumulation ◽  
Protein Accumulation ◽  
Clinical Settings ◽  
Bone Marrow Blast ◽  
Protein Levels ◽  
Small Cohort ◽  
Pre Treatment

Abstract Background: Activenuclear-cytoplasmic shuttling of proteins and RNAs, such as heterogeneous ribonucleoproteins (hnRNPs), is essential for the normal function and survival of eukaryotic cells and tumorigenesis (Dreyfuss et al. 1993 Annu Rev Biochem 62, 289; Gorlich and Mattaj 1996 Science 271, 1513). Up-regulation of exportin 1 (XPO1)/chromosomal maintenance 1 (CRM1), a member of the karyopherin-β family of nuclear export receptor proteins, has been implicated in solid and hematologic malignancies (Kau Kau et al. 2004).Selinexor (KPT-330) has been shown to be able block in vitro and in vivo XPO1/CRM1 functions and is currently in phase-II/IIb clinical trials for treatment of hematologic and solid tumors (Senapedis et al., 2014 Nat Rev Cancer 4, 106). However, the mechanisms underlying the selectivity and efficacy of selinexor are incompletely understood, and no biomarkers are currently available to predict clinical responses to selinexor in clinical settings. In this study, we focus on determining the effects of selinexor on the nuclear-cytoplasmic shuttling of hnRNPs, particularly hnRNPK and hnRNPA1, to elucidate the roles of the hnRNPs in the regulation of selectivity and efficacy of selinexor in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Method:We performed growth inhibition/killing assays, histopathologic evaluations, immunohistochemical studies, subcellular fraction western blotting, super-resolution stimulated emission depletion (STED) confocal microcopy and siRNA knockdown experiments. Results: Our in vitro experiments demonstrate a marked increase in XPO1/CRM1 protein and decrease in TP53 in our azacitidine-resistant MDS/AML cell lines compared to our azacitidine-sensitive MDS/AML cell lines. Selinexor treatment efficiently blocks export of hnRNP K from nuclei and increased nuclear accumulation of hnRNPK and inhibits MDS/AML cell growth, while the protein levels of XPO1/CRM1 and TP53 remain unchanged. Our experiments using clinical bone marrow specimens show no significant difference in the total protein level or nuclear accumulation of XPO1/CRM1 between the normal control and MDS or AML bone marrow specimens. In contrast, a strong positive correlation between MDS/AML disease progression and hnRNPK protein accumulation is observed in those clinical specimens. We have extended our experiments to clinical bone marrow specimens from a small cohort in a clinical trial for selinexor in AML at the University of Chicago (NCT02573363). In our small cohort, 5 patients responded to selinexor, 4 patients did not respond and 1 had a partial response. All 5 responders show a striking decrease in their bone marrow blast percentage from their pre-treatment marrows (average blast percentage 37.4%) to their post-treatment (average blast percentage 1.8%). Non-responders show no such difference in pre and post-treatment blast percentage (56.3 and 57.1%, respectively). Importantly, our experiments demonstrate a marked difference in the protein accumulation and subcellular localization of hnRNPK and hnRNPA1, another member of the hnRNP family, between selinexor-responder and selinexor-non-responder bone marrow specimens. Specifically, selinexor responders had much higher levels of hnRNPK and hnRNPA1 proteins in their pre-treatment bone marrows than non-responders, despite the fact that the latter had higher bone marrow blast percentages on average. There is markedly reduced accumulation of hnRNPK and hnRNPA1 in the post-selinexor treatment bone marrow specimens from the responders, but not the non-responders, suggesting these hnRNPs as key therapeutic targets for selinexor in MDS and AML. In contrast, no significant change in XPO1/CRM1 protein levels is observed in the selinexor-responder vs. selinexor-non-responder bone marrow specimens. Conclusion:Our data have revealed a novel drug-action mechanism by which selinexor impairs the nuclear-cytoplasmic shuttling of hnRNPK and hnRNPA1 in MDS and AML cells. Differential expression and localization of these hnRNPs in normal vs. MDS vs. AML cells may provide the rationale for the preferential killing of leukemia cells by selinexor. Our data also suggest the possibility to develop novel hnRNP-based biomarkers to predict the response to selinexor in clinical settings. Disclosures Liu: Karyopharm: Research Funding; BMS: Research Funding.

GSK1120212, a MEK1/MEK2 Inhibitor, Demonstrates Acceptable Tolerability and Preliminary Activity In a Dose Rising Trial In Subjects with AML and Other Hematologic Malignancies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3281-3281 ◽  
Author(s):  
Gautam Borthakur ◽  
James M. Foran ◽  
Tapan Kadia ◽  
Elias Jabbour ◽  
Paul Wissel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phase I ◽  
Cell Lines ◽  
Research Funding ◽  
Loading Dose ◽  
Bone Marrow Blast ◽  
Blast Count ◽  
Visual Changes ◽  
Refractory Aml

Abstract Abstract 3281 Background: MEK (mitogen-activated extracellular signal-related kinase) is downstream of the RAS/RAF pathway and is activated by many upstream oncogenic drivers. GSK1120212 is a potent and selective allosteric inhibitor of MEK 1 and 2 kinases. In vitro, GSK1120212 inhibited proliferation of myeloid cell lines selectively as compared to lymphoid cell lines. This 2 part study of a single daily oral dosing regimen was conducted to define the recommended Phase 2 dose, evaluate pharmacokinetics, and assess preliminary activity in patients with relapsed or refractory AML, MDS, ALL or CMML. Methods: Subjects with WBC< 30,000/uL who met hepatic, renal and cardiac function criteria were eligible for participation. GSK1120212 was given orally, once daily in the following dose cohorts: 3mg loading dose followed by 1mg/day (n=3), 1mg/day without loading dose (n=1), and 2mg/day without loading dose (n=9). The loading dose was discontinued after cohort 1, based on findings in the phase I solid tumor study (J Clin Oncol 28:15s, 2010 (suppl, abstr 2503). Results: Fourteen subjects (10 with AML, 2 MDS transformed to AML, 1 MDS, 1 ALL) entered the trial. Eight were male, and median age was 65 years (range 33 to 85). Pharmacokinetic analysis showed that, upon repeat dosing, GSK1120212 exposure increased in a dose-proportional manner, had a small peak:trough ratio of approximately 4 and an effective half-life of approximately 7.7 days. Steady state concentrations were reached by day 15. Both single and repeat dose pharmacokinetics of GSK1120212 appeared to be similar to a phase I study in patients with solid tumors. Systemic exposure exceeded concentrations that inhibited in vitro leukemic cell proliferation. At the 2mg/day dose level (n=9), drug-related adverse events were diarrhea (7 overall; 6-Grade 1/2, 1-Grade 3), rash (3-Grade 1/2), fatigue (4-Grade 1/2), visual changes (3 Grade 1/2). One subject experienced a Grade 2 reversible serous retinopathy associated visual changes which resolved after drug discontinuation. One dose limiting toxicity was seen in a subject with disease-related Grade 4 thrombocytopenia and pneumonia who experienced a Grade 5 cerebrovascular accident possibly related to drug. One subject in the 2mg/day cohort achieved a CR; peripheral blast count was reduced from 30% at baseline to 0% and bone marrow blast count was reduced from 50% at baseline to 3%. During this time, platelet count increased from 48K to a maximum of 276K. Initial salutary effect was seen after 2 weeks on therapy and duration of CR was 4 weeks bone marrow blast count was 3% and 5%, respectively, at the beginning and end of the 4 week CR duration. Conclusion: GSK1120212 administered at 2mg/day orally was tolerable in subjects with relapsed or refractory AML and other leukemias. This dose regimen achieved plasma concentrations sufficient for target inhibition and showed preliminary anti-leukemic clinical activity. Based on these results, a phase II study in AML, MDS and CMML has been initiated. Disclosures: Borthakur: GlaxoSmithKline: Research Funding. Foran:GlaxoSmithKline: Research Funding. Kadia:GlaxoSmithKline: Research Funding. Jabbour:GlaxoSmithKline: Research Funding. Wissel:GlaxoSmithKline: Employment. Cox:GlaxoSmithKline: Employment. Xu:GlaxoSmithKline: Employment. Bauman:GlaxoSmithKline: Employment. Baccus:GlaxoSmithKline: Research Funding. Connor:GlaxoSmithKline: Research Funding. Cortes:GlaxoSmithKline: Research Funding. Kantarjian:GlaxoSmithKline: Research Funding.

Correlates of Lenalidomide Induced Immune Stimulation and Response in CLL: Analysis in Patients on Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 979-979 ◽  
Author(s):  
Georg Aue ◽  
Stefania Pittaluga ◽  
Delong Liu ◽  
Larry Stennett ◽  
Susan Soto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Expression Profiling ◽  
Research Funding ◽  
Immune Stimulation ◽  
Intramural Research Program ◽  
Pre Treatment ◽  
Program Research

Abstract Abstract 979 Lenalidomide's mechanism of action in chronic lymphocytic leukemia (CLL) is not well understood. In vitro data suggest that anti-leukemic immune responses are important. Tumor flare reactions during treatment have been associated with response in some but not other studies. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/refractory CLL (clinicaltrials.gov: NCT00465127). Here we report final clinical data and results of multiple translational analyses that indicate that an IFNy centered immune response is critical for response. A 3 week on, 3 weeks off treatment scheme (42 day cycles) was chosen to pulse immune stimulation while trying to minimize myelosuppression. The starting dose was 20 mg daily for the first 10 patients and 10 mg for the subsequent 23. Response was measured at 24 weeks. 5 patients, 4 with del 17p, achieved a PR by IWCLL criteria (16%) and were eligible to continue drug for 4 more cycles; the PFS in these patients was 16 months compared to 7 months for all other (p<0.001). Myelosupression remained the limiting side effect. A cytokine release syndrome often accompanied by tumor flare reactions was seen in 78% of patients in cycle 1 and often recurred in subsequent cycles. Compared to other studies it appears that the long treatment free period increased the inflammatory reaction upon restarting of L. All correlative analyses reported here were performed on PBMCs, lymph node (LN) core biopsies and serum obtained from patients during cycle 1 and 2 and included flow cytometry, gene expression profiling (Affymetrix arrays), and cytokine measurements. Nine patients with decreased lymphadenopathy ≥10% (10–85%) on CT after 4 cycles were considered responders (R) for correlative studies. There was a significant decrease in CLL count (median 14% on day 8 and 49% on day 22, p<0.01) and in the number of circulating T (CD3, CD4, CD8) and NK-cells (n=22, p<0.05) with no difference between R and non-responders (NR). In contrast, the CD3 count in LN core biopsies increased 1.4 fold in R compared to matched pre-treatment biopsies (p<0.05) with no change in NR (0.95 fold). In the L free interval CLL cells rebounded to pre-treatment levels. A rapid rebound of CLL counts during treatment interruptions has been previously described but its mechanism is not well understood. In migration assays we observed a 3-fold increased migration towards SDF-1 for L compared to control cells (p=0.03), indicating that increased homing of lymphocytes to tissue sites may be responsible for the rapid decrease in peripheral counts. The cell surface molecules CD40, 54, 86, 95, DR5 were upregulated (p<0.05) while CD5 and 20 were downregulated (p<0.001) on circulating CLL cells. Effects on CD54 and CD5 were stronger in R than NR (p<0.05). Next we performed gene expression profiling on purified PB-CLL cells and LN core biopsies obtained on day 8. L induced upregulation of 95 genes, many of which are known to be regulated by interferon gamma (IFNγ). The comparison with a gene expression signature induced by recombinant IFNγ in CLL cells cultured in vitro confirmed the significant induction of a typical IFNγ response by L in vivo (n=24, p<0.0001). The IFNγ response in PB-CLL cells was no different in R vs NR (n=12, p=0.78), but in LN biopsies it was more prominent in R (n=7) than NR (n=5) (p<0.05). Consistently the IFNG gene was upregulated in LN biopsies of R but actually decreased in NR (p=0.001). Serum IFNγ levels were elevated on L (n=14 at all time points, day 4 p=0.03, day 8 p=0.01, day 22 p=0.02, day 49 p<0.01), but off drug returned to pretreatment levels. Next we sought to determine the source of IFNγ. The tumor cells are ruled out as IFNG was not expressed in purified CLL cells. By flow cytometry the number of IFNγ secreting CD4 T-cells increased on day 8 from 0.8% to 1.5%, p=0.006), an effect that was stronger in R had than NR (p<0.05). IFNγ positive NK cells did not increase on L. These data provide a first mechanistic link between the degree of Lenalidomide induced immune activation to clinical response in CLL. Based on our experience we suggest that continued dosing of L may be superior to dose interruptions. Disclosures: Aue: NHLBI, Intramural Research Program: Research Funding. Off Label Use: Lenalidomide is not FDA approved for CLL. Wiestner:NHLBI, Intramural Research Program: Research Funding.

Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 143-143 ◽  
Author(s):  
Saar Gill ◽  
Sarah K Tasian ◽  
Marco Ruella ◽  
Olga Shestova ◽  
Yong Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Research Funding ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1297-1297 ◽  
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.

Microrna-Dependent Modulation Of Osteogenesis In a 3D In Vitro Bone Marrow Model System Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3093-3093
Author(s):  
Michaela R Reagan ◽  
Yuji Mishima ◽  
Yong Zhang ◽  
Patricia Maiso ◽  
Salomon Manier ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Research Funding ◽  
3D Model ◽  
Human Cancer ◽  
Model System ◽  
Advisory Board ◽  
Alizarin Red ◽  
Non Destructive

Abstract Introduction Recent evidence indicates that tumor cells are not only influenced by their microenvironment, but are also able to drastically alter their surroundings leading to cancer progression. Multiple Myeloma (MM) involves clonal proliferation of malignant plasma cells within the bone marrow, inhibition of osteoblast function, and increased osteoclast activity leading to osteolytic lesions. Our work aims to understand the bi-directional interactions between MM cells and mesenchymal stromal cells (MSCs), using both 2D and 3D in vitro co-culture bone marrow models. Methods We developed a 3D in vitro model system to better mimic myeloma growth within the bone marrow using human MSCs (hMSCs) and fluorescent-, luciferase-labeled MM cell lines seeded into porous, autofluorescent silk scaffolds. Proliferation and osteogenic differentiation of myeloma patient (MM-) and normal donor (ND-) MSCs cultured with or without MM.1S cells were characterized in 2D culture and 3D scaffolds. Non-destructive bioluminescent imaging and fluorescent confocal imaging were used to observe cell growth and cell-cell interactions within scaffolds. Histology was performed to confirm changes in extracellular matrix (ECM) production and bone tissue formation. microRNA (miRNA) profiling was performed on primary ND- (n=3) and MM-MSCs (n=7) using Nanostring technologies. We analyzed 800 human miRNAs from miRBase v.18 and 230 human cancer-related genes using the nCounter® Human Cancer Reference Kit. Gain-of function studies (miRvana mimics) were performed for miRNAs that were down-modulated in MM vs ND-MSCs, and in the 3D model MSCs co-cultured with MM.1S vs MSCs alone, using lipofectamine. Modulation of osteogenesis was evaluated using alizarin red staining and qRT-PCR for the osteogenic markers: IBSP (integrin-binding sialoprotein), Col1a1 (collagen, type I, alpha 1), RUNX2 (runt related transcription factor 2), ALPL (alkaline phosphatase), OPN (secreted phosphoprotein 1), and BGLAP (bone gamma-carboxyglutamate (gla) protein). Results MM-MSCs presented with a lower proliferation rate compared to ND-MSCs and this phenotype was also observed in ND-MSCs co-cultured in the presence of MM.1S cells compared to ND-MSCs alone. Moreover, significant inhibition of MSC growth was evident when co-cultured with MM.1S cells, using a 3D model (Figure 1), where inhibition of osteogenesis, and ECM production were also documented. Alizarin red staining demonstrated inhibited ability for MM-MSCs to undergo osteogenic differentiation. In addition, MM-MSCs differed from ND-MSCs at the gene and miRNA level. Specifically, CDKN1A and CDKN2A were over-expressed in MM vs. ND-MSCs, (P<0.05; fold change >1.2), thus explaining, at least in part, the decreased proliferation of MM-MSCs vs ND-MSCs. Moreover, down-regulation of specific miRNAs (miRNA-199a, -24-3p, -199a, -15a-5p, -16-5p) was demonstrated in MM- vs ND-MSCs, as well as in ND-MSCs vs ND-MSCs co-cultured with MM.1S, using the 3D model. By over-expressing miRNA-199a, -15a-5p and -16-5p, we were able to increase the osteogenic potential, thus suggesting their role in modulating osteogenesis in MM-MSCs. Conclusions Our 3D platform provides a simple, non-destructive, flexible, and clinically relevant tool to spatially and temporally model myeloma growth within bone. It recapitulates decreased bone formation as seen in MM patients and suggests miR-199a-3p, 15a-5p and 16-5p as novel bone anabolic targets. Disclosures: Tai: Onyx: Consultancy. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Mobilization Of Blasts and Leukemia Stem Cells by Anti-CXCR4 Antibody BMS-936564 (MDX 1338) in Patients With Relapsed/Refractory Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3882-3882 ◽  
Author(s):  
Sylvia Chien ◽  
Lauren E. Beyerle ◽  
Brent L. Wood ◽  
Elihu H. Estey ◽  
Frederick R. Appelbaum ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Bone Marrow ◽  
Research Funding ◽  
Annexin V ◽  
Fold Increase ◽  
Cxcr4 Expression ◽  
Leukemia Stem Cells ◽  
Pre Treatment ◽  
Refractory Aml ◽  
Peak Value

Abstract Background Bone marrow homing of AML is dependent on CXCR4, and high levels of CXCR4 expression correlate with worse survival in AML (Rombouts et al 2004, Spoo et al 2007). CXCR4 antagonists overcome environment adhesion mediated drug resistance and enhance chemotherapy induced cytotoxicity (Liesveld et al 2007, Zeng et al 2009, Nervi et al 2009, Beider et al 2010). Plerixafor, a small molecular CXCR4 inhibitor, was studied in a phase I trial of newly diagnosed AML patients (Uy et al ASH 2011), and BMS-936564, a fully-human monoclonal antibody to CXCR4, in combination with MEC (mitoxantrone, etoposide, cytarabine), is currently under study in relapsed/refractory AML. Method The clinical trial completed a phase I dose escalation phase in AML patients, with increasing concentrations of BMS-936564 in 4 dose cohorts (0.3,1,3, and 10 mg/kg) and is currently enrolling a cohort of first salvage AML patients at the maximum dose of 10 mg/kg. The initial cohort of patients at 0.3 mg/kg received three weekly doses of antibody on days 1, 8, 15 [monotherapy period of cycle 1 (21 days)], followed by the same dose of antibody on days 1, 8, 15 of cycle 2 plus MEC chemotherapy [days 1-5 of cycle 2, (28 day cycle)]. After enrollment of the first cohort, the protocol was amended to reduce the monotherapy period to 1 week (1 dose of BMS-936564) in cohorts 1, 3, and 10 mg/kg, followed by the same combination regimen. As a companion study to this trial with the anti-CXCR4 antibody, we are investigating CXCR4 expression, timing of mobilization of leukemic blasts and leukemia stem cells (LSCs), and induction of apoptosis. Mobililzation of LSCs will be critical to eradication of leukemia, as they might serve as a reservoir for drug resistance and future relapse. We analyzed serial blast and LSC populations from blood and bone marrow samples from patients undergoing treatment by flow cytometry for phenotype, CXCR4 and annexin V expression. The putative LSCs were defined as CD34+CD38-CD123+ or by aldehyde dehydrogenase. Results An independent assessment of CXCR4 expression in 56 consecutive AML patients from our institution not related to this clinical trial revealed a mean % expression of 31%, range 1-99%, with mean fluorescence intensity (MFI) of 2092, range 319-7942. A sample of 18 patients showed a correlation in CXCR4 expression between gated blasts derived from blood and bone marrow samples from the same patient (For % expression, r2=0.85, p=5e-8; MFI r2=0.45, p=0.002). Our site has enrolled 24 AML patients thus far on the above noted trial of BMS-936564. Administration of BMS-936564 resulted in brisk mobilization of leukemic blasts in 14/24 patients that initially peaked within at 2-6 hours post start of infusion in most patients, with an average of 2.1-fold increase ± 1.8 fold (range 1.06-8.96 fold), and some blasts continued to be in circulation for days. In most cases, the samples for which mobilization was not observed either did not have circulating blasts at baseline, or were from patients who received lower doses of BMS-936564. In addition, CD34+CD38-CD123+LSCs were also mobilized post-treatment with BMS-936564, and in some cases, continued to rise over the subsequent days, during which the blast population declined. The average rise in %CXCR4 was from 29.3% pre-treatment to 69.8% peak value for blasts, and 23.0% pre-treatment to 75.6% peak value for LSCs. Although a direct correlation between CXCR4 expression by blasts and fold mobilization was not apparent, the highest fold increase in mobilization (∼9-fold) did occur in the patient with a moderately high level of CXCR4 expression, 42.5%. In this patient, there was also a sharp decline in circulating CXCR4 positive cells within 2 days and the patient achieved complete remission. BMS-936564 has demonstrated apoptosis in some preclinical models (Kuhne MR et al Clin Cancer Res v19(2): 357-66 (2013)).) For most patients, there was some increase in annexin V staining observed during the first 96 hours after antibody exposure. One patient sample with initial low baseline level of apoptosis exhibited a rise in annexin V staining (from 6% to 48%) that peaked on day 3 after administration of BMS-936564. Conclusion These data demonstrate that BMS-936564 induces mobilization of both AML blasts and LSCs, which may enhance chemotherapy-induced cytotoxicity in relapsed/refractory AML. Disclosures: Chien: Bristol-Myers Squibb: Research Funding. Cardarelli:BMS: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Becker:Bristol-Myers Squibb: Research Funding.

Chronic Lymphocytic Leukemia Cells With Trisomy 12 Home To The Bone Marrow In a CXCR4-Independent Manner and Are Prone To Proliferate In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 870-870
Author(s):  
Evelyn Hutterer ◽  
Elisabeth Hinterseer ◽  
Sylvia Ganghammer ◽  
Gabriele Brachtl ◽  
Daniela Asslaber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Dependent Manner ◽  
Independent Manner ◽  
Atypical Cell ◽  
Trisomy 12 ◽  
Inside Out

Abstract Trisomy 12 (tri12) is a frequent chromosomal aberration in chronic lymphocytic leukemia (CLL) associated with atypical cell morphology, high in vivo tumor proliferation activity and a predisposition to Richter’s transformation. Tri12 harboring CLL cells express increased levels of the negative prognostic marker CD49d, the α4 subunit of the integrin very late antigen 4 (VLA-4), which we previously identified as a key regulator of CLL cell homing to bone marrow (BM). During this process, inside-out activation of VLA-4 upon CXCR4 binding to endothelially displayed CXCL12 is thought to upregulate the adhesive properties of VLA-4 and augment the arrest of CLL cells on the VCAM-1 presenting vessels. Here, we investigated the functional interplay of VLA-4 and CXCR4 in CLL carrying tri12. We first found that the upregulation of CD49d expression in this subset (MFIR CD49d 9.8±5.3 (n=22) vs. 2.7±3.9 (n=126), p<0.0001) was paralleled by their reduced CXCR4 expression (MFIR CXCR4 11.8±7.2 (n=22) vs. 22.7±14.2 (n=126), p=0.0003). Using short term adoptive transfers, we compared the ability of tri12 and no tri12 CLL cells to home to the BM of NOD/SCID mice. 5-10x106 CLL cells were injected into tail vein and homing was evaluated after 3 hours. Based on their more frequent CD49d high phenotype, we observed increased homing rates (homed human CLL cells per 106 injected cells per 106 acquired murine cells) of tri12 compared to no tri12 CLL (225±160 (n=7) vs. 90±117 (n=20), p=0.025). However, when comparing CD49d+ tri12 and CD49d+ no tri12 subsets, we did not observe any significant differences in their homing capacity. To further study CXCL12/CXCR4 function in BM homing, we pretreated mice with either the novel CXCL12 antagonist NOX-A12 or the CXCR4 inhibitor AMD3100 prior to CLL cell injection. While homing of no tri12 CLL cells (n=3, in duplicates) was reduced by both pretreatments (homing rates 137 vs 38 vs 30), the homing capacity of tri12 CLL cells (n=3, in duplicates) was not affected. We next tested whether VLA-4 expressed on these cells was able to undergo CXCL12-induced activation and support cell arrest under shear conditions. To this end, we perfused CLL cells over VCAM-1 or VCAM-1/CXCL12 substrates and analyzed rates and categories of cell tethering at a single cell level by videomicroscopy. CXCL12 induced the arrests of no tri12 CLL cells (n=3) on VCAM-1 under shear flow in a CXCR4 and VLA-4 dependent manner. In contrast, tri12 CLL cells (n=3) robustly tethered to VCAM-1 in the absence of the chemokine, and interactions could not be further enhanced by additional CXCL12 nor could they be abrogated by use of AMD3100. This failure of CXCR4-induced adhesion was not based on a general defect in CXCR4 functionality as in vitro chemotaxis of tri12 CLL cells (n=5) towards CXCL12 was fully maintained. To detect potential differences in VLA-4 affinity regulation, we used a conformationally sensitive antibody that recognizes epitopes induced by VLA-4 ligation, and an LDV-containing VLA-4 specific ligand to probe resting integrin affinity. Also, we used a small fluorescent ligand to study rapid VLA-4 affinity changes during inside-out chemokine induced activation. On resting tri12 CLL, VLA-4 exhibited an affinity state similar to that observed on circulating lymphocytes, and tri12 CLL cells failed to undergo the rapid affinity up-regulation triggered by CXCL12 pretreatment, in keeping with tethering experiments. Next, we investigated whether the tumor microenvironment has a different influence on the behavior of the tri12 subset. Therefore we subjected the cells to in vitro co-cultures mimicking the lymphoid proliferation centers. Basal levels of the early activation marker CD69 were similar in tri12 CLL compared to no tri12 cases. Tri12 CLL, however, underwent stronger activation when cultured in presence of accessory cells (%CD69+ cells 60.0±18.5 (n=4) vs. 17.7±20.1 (n=19), p=0.008). Moreover, in several setups, proliferation rates of these cells were increased, irrespective of the proliferative stimulus and detection method used. In summary, our results provide a mechanistical basis at least in part explaining the peculiar and clinical features of the tri12 CLL subset. In light of the specific migratory and proliferative properties of tri12 cells and novel agents targeting particularly these functions, our findings may also imply therapeutical consequences. Disclosures: Greil: NOXXON Pharma AG: Research Funding. Hartmann:NOXXON Pharma AG: Research Funding.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 350-350
Author(s):  
Kyung-Hee Chang ◽  
Amitava Sengupta ◽  
Ramesh C Nayak ◽  
Angeles Duran ◽  
Sang Jun Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Negative Regulator ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

mTOR Kinase Inhibitors Enhance Efficacy of TKIs in Preclinical Models of Ph-like B-ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2763-2763 ◽  
Author(s):  
Moran Gotesman ◽  
Thanh-Trang T Vo ◽  
Sharmila Mallya ◽  
Qi Zhang ◽  
Ce Shi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Inhibitor Therapy ◽  
Cellular Viability ◽  
Murine Bone Marrow ◽  
Pdx Models

Abstract Background and Rationale: B-lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. While event-free survival (EFS) exceeds 85% for most patients treated with contemporary therapy, outcomes are very poor for children who relapse, highlighting a need for new treatments. In particular, children with Philadelphia chromosome-like (Ph-like) B-ALL (who lack BCR-ABL1 rearrangement) have high rates of relapse and mortality with conventional chemotherapy. Transcriptional profiling and genomic sequencing of Ph-like ALL specimens have identified a variety of alterations that activate oncogenic kinase signaling, including rearrangements (R) of CRLF2, ABL1, and PDGFRB. Addition of the tyrosine kinase inhibitor (TKI) imatinib to chemotherapy has dramatically improved EFS for patients with BCR-ABL1-rearranged (Ph+) B-ALL, and it is hypothesized that TKI addition to therapy will similarly improve outcomes for patients with Ph-like ALL. Our prior preclinical studies in Ph+ B-ALL demonstrated enhanced efficacy of combining TKIs (imatinib or dasatinib) with mTOR kinase inhibitors (TOR-KIs) (Janes et al., Nature Medicine 2010; Janes et al, Leukemia2013). In the current studies, we hypothesized that dual kinase inhibitor therapy would have superior anti-leukemia cytotoxicity in Ph-like ALL and thus investigated combined TKI and TOR-KI treatment using patient-derived xenograft (PDX) models of childhood Ph-like ALL. Methods: For in vitro studies, viably cryopreserved leukemia cells from established ABL1-R Ph-like ALL PDX models (2 ETV6-ABL1) were incubated with the TKI dasatinib, TOR-KIs, or both TKI + TOR-KI for 72 hours prior to flow cytometric assessment of cellular viability via Annexin V and propidium iodide staining. Two chemically distinct TOR-KIs (MLN0128 or AZD2014) were used to confirm on-target effects. Additional primary ABL1-R or PDGFRB-R Ph-like ALL specimens were plated in methylcellulose without or with inhibitors in colony-forming assays. Phosphoflow cytometry (PFC) analysis of ALL cells incubated with inhibitors was also performed to measure the ability of TKIs and TOR-KIs to inhibit intracellular ABL1 and PI3K/mTOR signaling pathways. For in vivo studies, Ph-like ALL PDX models were treated with dasatinib, the TOR-KI AZD8055, or both drugs via daily oral gavage for 8 days. Human CD19+ ALL was quantified in murine spleens and bone marrow at end of treatment with quantification of cycling cells by EdU incorporation. PFC analysis of murine bone marrow was also performed 2 hours after drugs were dosed, to measure in vivo inhibition of signaling proteins. Results: Combined in vitro treatment with dasatinib and MLN0128 or AZD2014 decreased cellular viability more than inhibitor monotherapy. Similarly, in a set of CRLF2-rearranged samples, mTOR inhibitors augmented killing by the JAK2 inhibitor BBT-594. Incubation of primary ABL1-R or PDGFRB-R ALL cells with both dasatinib and AZD2014 more robustly inhibited colony formation than did inhibitor monotherapy. In in vitro PFC analyses of ABL1-R samples, we observed expected dasatinib-induced inhibition of phosphorylated (p) STAT5. Inhibition of the mTOR substrate pS6 was observed with dasatinib, MLN0128, and AZD2014 with more complete inhibition achieved when dasatinib combined with either MLN0128 or AZD2014. Similarly, in vivo treatment of PDX models with dasatinib and AZD8055 reduced leukemia burden and pS6 signaling more completely than either inhibitor alone. Importantly, dual inhibition decreased the percentage of cycling human ALL cells in murine bone marrow, but preserved cycling in normal mouse bone marrow cells in the same animals. Our data thus provide additional compelling preclinical rationale for combined inhibitor therapy with TKIs and TOR-KIs in Ph-like ALL. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

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