Presence Of Stroma Research Articles

Are disseminated tumor cells in bone marrow and tumor-stroma ratio clinically applicable for patients undergoing surgical resection of primary colorectal cancer? The Leiden MRD study.

PurposeCurrent TNM staging does not appropriately identify high-risk colorectal cancer (CRC) patients. The aim of this study was to evaluate whether the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) and the presence of stroma in the primary tumor, i.e., the tumor-stroma ratio (TSR), in patients undergoing surgical resection of primary CRC provides information relevant for disease outcome.MethodsPatients with primary CRC (n = 125), consecutively admitted for curative resection between 2001 and 2007, were included in the study. All patients underwent BM aspiration before surgery. Detection of tumor cells was performed using immunocytochemical staining for cytokeratin (CK-ICC). The TSR was determined on diagnostic H&E stained sections of primary tumors.ResultsDTCs were detected in the BM of 23/125 patients (18 %). No association was found between BM status and overall survival (HR 0.97 (95 % CI 0.45–2.09), p = 0.93). Also, no significant difference was found in their 5-year survival rate (resp. 72 % and 68 % for BM-positive versus BM-negative patients). The TSR was found to be associated with a worse overall survival (HR 2.16, 95 % CI 1.02–4.57, p = 0.04) with 5-year survival rates of 84 % versus 62 % for stroma-low and stroma-high patients, respectively. No relation was found between the presence of DTCs and TSR.ConclusionsOur data indicate that the presence of DTCs in the BM of CRC patients is not associated with disease outcome. The TSR was, however, found to be associated with a worse overall survival, which indicates that for CRC the tumor microenvironment plays an important role in its behavior and prognosis.

Abstract 5102: Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment

Abstract We previously showed that the heparan sulfate proteoglycan, perlecan (PLN)/HSPG2, is deposited into the matrix of reactive stroma surrounding tumors, where it concentrates heparin binding growth factors (HBGFs) and cytokines at the tumor-stromal border both in primary prostatic tumors and bone metastatic lesions. Heparan sulfate (HS) modifying enzymes produced in tumor-associated stroma play key roles in the delivery of these perlecan-sequestered cancer, stromal and tumor macrophage-produced HBGFs and cytokines, serving to enhance tumorigenesis and recruit other bystander cells to sites of metastasis. Studies presented here focused on the production of secreted HS-modifying enzyme heparanase and the two extracellular sulfatases, SULF1 and SULF2, in the prostatic tumor microenvironment. Our studies showed that SULF1 and heparanase mRNA levels are elevated in prostate cancer (PCa) cells of the LNCaP lineage in the presence of stroma, indicating that both stroma and PCa cells can be sources of these enzymes. We are presently examining a number of PCa patient derived xenografts to determine the prevalence of SULF-1 and heparanase elevation in castrate resistant disease. To study function, we developed a modified 3D hyaluronate gel system that contains HS-bearing recombinant perlecan domain I (PlnDI) to retain and deliver HBGFs and cytokines to various PCa cells cultured with bone marrow stromal cells, osteoblasts, or tumor macrophages. We are using this system to study the expression and individual functions of heparanase and the two SULFs in promoting HBGF-dependent PCa progression in a physiologically relevant system. Overexpression and gene deletion techniques allow us to determine the role that heparanase or SULFs play in each relevant cell type present in the tumor microenvironment, and thus determine the need for co-targeting of both tumor and reactive stroma. (Supported by NIH P01 and CAPES). Citation Format: Fabio H. Brasil, Valeria Ferrer, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5102.

Growth and aggressiveness factors affecting Monilinia spp. survival peaches

Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analyzed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r=0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r=0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest.

Growth and aggressiveness factors affecting Monilinia spp. survival peaches

Brown rot of stone fruit is caused by three species of Monilinia, Monilinia laxa, M. fructigena, and M. fructicola. Eleven components of 20 different isolates of each of the three Monilinia species were analysed to determine distinct aggressiveness and growth characteristics among the three fungi. M. fructicola showed the greatest lesion diameter, and the lowest incubation and latency period on fruit postharvest, however isolates of M. fructigena exhibited less aggressiveness components. Five growth characteristics of M. fructicola could be used to distinguish M. fructicola from the other two species. The dendrogram generated from only the presence of sclerotia and lesion length on infected fruit separated the 60 isolates into two clusters (r=0.93). One cluster was composed of the M. laxa and M. fructigena isolates and the other cluster comprised the M. fructicola isolates. However, the dendrogram generated based on the presence of stromata and sclerotia in the same colony of the three species when they were grown on potato dextrose agar, and the lesion diameter on fruit infected with each species separated the 60 isolates into three clusters (r=0.81). Each cluster comprised the isolates of each of three Monilinia spp. We discussed the effect of M. fructicola growth and aggressiveness differences on the displacement of M. laxa and M. fructigena by M. fructicola recorded in Spanish peach orchards and their effect on brown rot at postharvest.

The Combination of the Novel Syk Inhibitor TAK659 with Decitabine Exerts Synergistic Cytotoxic Effects Against FLT3/ITD Mutated Acute Myeloid Leukemia Cells

Background: Effective treatment regimens for FLT3/ITD+ AML patients are lacking. Recent data on the effects of FLT3 tyrosine kinase inhibition showed promising clinical activity, but outcomes have been stunted by drug resistance. Targeting additional signaling pathways besides FLT3 might therefore offer therapeutic benefit. Spleen tyrosine kinase (syk) is frequently overexpressed and constitutively activated in primary AML FLT3/ITD+ blasts. Pharmacologic inhibition of syk has been associated with antileukemic potential, consisting of inhibition of cell growth and induction of differentiation in AML cells. Hypomethylating agents (DNMTis) such as Decitabine (DEC) have emerged as attractive agents in the treatment of AML, partially due to their proapoptotic and differentiation inducing effects. There is a strong rationale for combining FLT3- and syk- inhibitors with DNMTis for FLT3/ITD+ AML. Here, we investigated whether the antileukemic effects of the novel, dual FLT3/syk inhibitor TAK659 (TAK) can be enhanced by the co-administration of DEC.Methods: Molm14 (M14) cells were incubated in culture medium in the presence and absence of stromal cells (MS5) for 72 hours. TAK or DEC was added in single agent or combined mode at the indicated concentrations. After the incubation period, cell growth was determined by MTT assays. Apoptosis, autophagy and cell cycle status were assessed by FACS analysis. FLT3- and syk- phosphorylation levels were detected by western blotting. Differentiation was assessed by morphology. A combination index (CI) was calculated using Calcusyn Software.Results: TAK exerted dose dependent effects against M14 cells. However, the presence of stromal cells significantly blunted TAK mediated cytotoxicity (50nM: 61.9±1.3% [off stroma] vs. 10.9±3.1% [on stroma], p<.001; 500nM: 91.2±0.5% [off stroma] vs. 17.4±2.5% [on stroma], p<.001, n=3-9; IC50: 40.6nM [off stroma] vs. 3.9µM [on stroma]). Although less marked when stroma was present, TAK combined with DEC yielded synergistically enhanced growth inhibition (off stroma: 50.2±3.9% [TAK659 50nM], 58.1±3.0% [DEC 1µM], 76.7±3.1% [TAK659 50nM+DEC 1µM; CI=0.77]; on stroma: 9.5±3.0%[TAK659 50nM], 42.9±3.1% [DEC1µM], 57.0±5.2%[TAK659 50nM+DEC 1µM; CI=0.62]) compared to single agent treatment (off stroma: p<.001 vs. TAK, p< .001 vs. DEC, on stroma: p<.001 vs.TAK, p=.03 vs. DEC, n=3). In line with these findings, combined treatment yielded greater levels of FLT3- and syk- dephosphorylation compared to single agent treatment both when stroma was present and absent. When compared to untreated controls (11.6±0.7%), Annexin V/PI staining (n=3) showed that induction of apoptosis was increased with TAK (18.9±3.9%; p=NS), DEC (39.2±4.8%; p<.01), and greatest when the two drugs were combined (44.2±6.7%; p<.01) in the absence stoma. In the presence of stroma, single agent DEC, and when combined with TAK, increased the fraction of apoptotic cells by up to two fold whereas TAK did not induce any meaningful apoptosis. Consistent with the induction of cell death, cell cycle analysis demonstrated the greatest increase in subG0 phase when TAK and DEC were combined. Both in the presence and absence of stroma, autophagic flux was increased when M14 cells were treated with DEC, and when DEC was combined with TAK whereas no difference was observed with TAK alone. The combination of TAK and DEC led to changes consistent with differentiation, as shown by more abundant cytoplasm and nuclei with deep indentations, folds and lobulation compared to untreated cells. DEC treated cells also showed signs of differentiation although these changes were less marked. No evidence of differentiation was observed in M14 cells when treated with TAK alone.Conclusions: 1. Stroma conferred protective effects against all treatments. 2. TAK exerted dose dependent cytotoxicity against M14 cells in the nanomolar to low micromolar range in the absence and presence of stroma, respectively. 3. The cytotoxic effects of TAK were enhanced when TAK was combined with DEC. 4. The antileukemic effects of TAK combined with DEC are multifaceted and include inhibition of cell growth, induction of apoptosis, autophagy and differentiation. 5. The combination of TAK and DEC represents a potentially important novel approach for FLT3/ITD+ AML patients. 6. Clinical trials incorporating syk-inhibitors into standard AML treatment protocols are currently under way. DisclosuresDos Santos:Amgen: Employment.

A Novel Three-Dimensional Co-Culture System to Study Leukemia in the Bone Marrow Microenvironment

The need for novel therapies in acute leukemia has been motivated by sub-optimal 5-year survival rates of 25.4% in acute myeloid leukemia (AML) and approximately 70% in acute lymphoblastic leukemia (ALL). While these rates are higher in the pediatric population, novel approaches are necessary in all age groups to improve outcomes.Pre-clinical studies of novel therapeutics using in vitro and in vivo methods remain suboptimal with frequent lack of correlation with clinical outcomes at the bedside. Recent evidence has shown that human leukemia xenografts into immunodeficient mice yield variable results, indicating that treatment using these methods is not replicable. When using in vitro cell culture methods, the well-documented protective effects of the bone marrow (BM) microenvironment (BMME) on leukemia are not mimicked. Furthermore, these techniques cannot be used to investigate the effects of novel agents on leukemia stem cells (LSC) and their mobilization, which is important in the ablation of leukemia. Thus, we explored a novel 3-dimensional co-culture system to study the effects of drugs on leukemia cells in the presence of stroma in an environment more similar to that of human leukemia in the BMME.We generated a 3-dimensional (3D) spheroid co-culture system using human stromal cell line (HS-5) cells or human mesenchymal stromal cells (hBMSC) from primary AML or ALL BM. To evaluate the dynamics of the 3D system, we labeled the stroma cells with GFP and the leukemia cells with mCherry. We observed rapid homing to the center of the 3D stroma. We evaluated ROS levels, proliferation status, hypoxia and expression of key niche proteins such as CXCL12 in leukemia cells found outside and inside the 3D system. These methods were compared to similar treatments in leukemia cell monolayer culture and 2-dimensional co-culture systems.We treated this system with various drugs such as cytarabine, doxorubicin, TG02 (a multi-kinase inhibitor with LSC mobilization effects), and plerixafor; we then harvested cells from the outer and inner layers and evaluated these separately by multi-parameter flow cytometry for viability and mobilization of LSCs in relation to the stroma and xenotransplant assays.Our studies reveal that the 3D culture system has lower ROS internally, suggesting a similarly hypoxic environment to BMME. Our studies also reveal that, when treated with cytarabine, AML cells closest to the stromal center of the spheroid remain protected, with higher viability compared to those farther from stroma, and even more so than leukemia cells in a 2-D bilayer with stroma or in a monolayer. A lower CXCL12 level was also observed in the stroma of leukemic BM compared to healthy BM within the co-culture system.This culture method possesses many of the characteristics of leukemia cells within the bone marrow niche and should be considered for future in vitro pre-clinical drug testing to model the tumor within its microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Azacitidine in Combination with the Selective FLT3 Kinase Inhibitor Crenolanib Effectively Disrupts Stromal Protection of CD34+ Leukemia-Initiating Cells (LIC) in FLT3-ITD+ Acute Myeloid Leukemia (AML)

Background: Effectively targeting the oncogenic mutation FLT3-ITD remains a crucial goal in acute myeloid leukemia (AML) therapy. Thus far, tyrosine kinase inhibitors (TKI) have not been able to eradicate the earliest leukemia-initiating cells (LIC) in FLT3-ITD+ AML, which are thought to be responsible for the frequent relapses seen in this disease. We have previously shown that LIC in FLT3-ITD+ AML persist during treatment with first-generation TKI despite effective inhibition of FLT3 phosphorylation owing to their selective protection by niche cells (Parmar et al, Cancer Res 2011). Hence, novel strategies to disrupt the protective interaction of stroma with FLT3-ITD+ LIC are urgently needed. Here, we asked whether stromal resistance of FLT3-ITD+ LIC can be overcome by the next generation TKI crenolanib alone or in combination with the hypomethylating agent azacitidine (AZA).Methods: The efficacy of crenolanib alone or in combination with AZA was assessed in the human FLT3-ITD+ cell lines MV4-11 and MOLM13 and FLT3-ITD transfected BaF3 cells as well as in primary human FLT3-ITD+ AML bone marrow samples. Cells were cultured in suspension or on the mesenchymal stromal cell line EL08-1D2, which mimics the bone marrow niche and maintains LIC in vitro (Parmar et al, Cancer Res 2011). Cultures were treated with DMSO, crenolanib and/or AZA for defined periods. Apoptosis, cell cycle and differentiation of AML cells were analyzed by flow cytometry. Clonogenic capacity and frequency of primitive FLT3-ITD+ stem/progenitors were probed by standard CFU and LTC assays. Engraftment potential of FLT3-ITD+ patient-derived xenograft (PDX) AML cells after treatment with crenolani, AZA or the combination thereof was assessed in the NSG xenograft model (Vick et al, PlosOne 2015). Treatment-induced alterations in FLT3-ITD downstream signaling were investigated by western blots.Results: Crenolanib effectively inhibited FLT3 and STAT5 phosphorylation in FLT3-ITD+ cells in suspension as well as in EL08-1D2 supported co-cultures whereas AZA had no effect on FLT3 signaling pathways. Monotherapy with crenolanib but not AZA effectively induced apoptosis and inhibited growth of FLT3-ITD+ cell lines. Primary CD34+ FLT3-ITD+ progenitor cells were also highly susceptible to inhibition by crenolanib as a single agent. However, crenolanib was completely unable to eradicate primitive CD34+ FLT3-ITD+ LIC when protected by niche cells as assessed by CFU and LTC assays as well as xenotransplantation into NSG mice. In contrast, the combination of crenolanib and AZA resulted in efficient apoptosis and dramatically impaired clonogenic capacity of FLT3-ITD+ LIC even in the presence of stroma. Pretreatment of EL08-1D2 cells with AZA before co-culture with AML cells did not influence stromal protection against crenolanib. Furthermore, soluble stromal factors did not account for TKI resistance. Successful targeting of stromally protected FLT3-ITD+ LIC by the combination of AZA and crenolanib is currently being confirmed by xenotransplantation in NSG mice.Conclusion: The combination of azacitidine and the selective next generation FLT3 kinase inhibitor crenolanib is a novel promising treatment regimen to overcome niche protection and selectively target LIC in FLT3-ITD+ AML. We hypothesize that the mechanism of action involves loss of quiescence in FLT3-ITD+ LIC leading to increased susceptibility towards TKI as well as induction of differentiation by AZA. In depth analysis of involved signaling pathways and phenotypic alterations in LIC are ongoing. DisclosuresKeller:Pfizer: Consultancy; Roche: Consultancy, Honoraria. Götze:Novartis: Honoraria; Celgene Corp.: Honoraria.

Defining Primary Marrow Microenvironment-Induced Synthetic Lethality and Resistance for 2,684 Approved Drugs Across Molecularly Distinct Forms of Multiple Myeloma

▪Multiple Myeloma (MM) is a prototypical neoplasm for the study of tumor-microenvironment interactions and influences on drug response. These interactions within the bone marrow (BM) alter the signaling state of MM cells and their relative dependence on pharmacological targets. Conversely, many efforts to identify and validate drug targets in MM are conducted outside of this context. This raises the possibility that systematic re-evaluation of the current pharmacopeia may identify drugs with previously unappreciated capacity for targeting MM cells within the marrow environment.To this end, we utilized the compartment-specific bioluminescence platform (CS-BLI) to characterize the activity of 2,684 FDA-approved drugs from The Johns Hopkins Drug Library (JHDL) in three distinct MM subtypes, in the presence or absence of patient-derived CD138-negative bone marrow stromal cells (BMSCs). Anti-MM activity was evaluated at 100 nM concentrations over 72 h in MM1S (t(14;16), KRASG12A, TRAF3LOF), L363 (t(20;22), NRASQ61H, p53S261T), and OPM2 (t(4;14), FGFRK560E, p53R175H) lines. These lines demonstrate phenotypes of strong, medium, and low BMSC-induced growth enhancement, respectively. Active drugs were placed into 4 categories: type 1 - having potent anti-MM activity independent of BMSC interactions (no stromal effect), type 2 - having anti-MM activity only in the presence of BMSCs (stroma-dependent "synthetic lethality"), type 3 - having anti-MM activity that is decreased in the context of BMSCs (stroma-dependent resistance), and type 4 - otherwise inactive agents that demonstrate pro-MM activity in context of BMSCs (stroma-dependent stimulants).In this study, for MM1S, L363, and OPM2, respectively, we identified 103, 118, and 108 type 1 drugs, 217, 105, and 76 type 2 drugs, 128, 75, and 16 type 3 drugs, and 124, 33, and 38 type 4 drugs. For each category of drug phenotype, we assessed overlap across the three MM cell lines. We observed high degree of overlap for type 1 drugs (67 drugs active in all three models), while more diversity between lines was evident across the 3 lines for type 2-4 drugs, whose activity is altered by interaction with BMSCs (Figure 1). Specifically, focusing on agents demonstrating BMSC-associated stimulation, adrenergic drugs consistently stimulated MM growth in context of BMSCs, while glucocorticoids consistently grouped as type 3 agents (demonstrating BMSC-associated resistance). Interestingly, carfilzomib was also subject to BMSC-associated resistance.Despite differences in drugs demonstrating stroma-induced lethality across the MM cell lines, salicylates were commonly represented in this category. In addition to the salicylates, tofacitinib, a Janus kinase (JAK) inhibitor, demonstrated a strong capacity to elicit a stroma-dependent synthetic lethal phenotype and ruxolitinib, another inhibitor in the same class, showed a similar, yet distinct pattern of stroma-mediated sensitization.In conjunction with our screen, we performed an RNA-seq analysis to assess differential gene expression between MM in monoculture vs. in co-culture with BMSCs. Expression analysis revealed 4.0 fold increase in JAK3 expression induced by co-culture with primary BMSCs, as well as induction of a STAT3 transcription factor fingerprint by ChIP-seq enrichment analysis. A detailed dose-response analysis of tofacitinib revealed no anti-MM activity against MM cells in isolation at physiological concentrations, but showed typical sigmoidal log-dose response dynamics in the presence of stroma and a dynamic range that completely abrogated the growth advantage attributable to stromal stimulation.This phenomenon of BMSC-dependent pharmacology identifies tofacitinib as an intriguing candidate for repurposing as an agent demonstrating stroma-induced synthetic lethality against MM. Further evaluation of this agent in combination with other anti-MM agents, like bortezomib, is also warranted. Taken together, this study demonstrates specific anti-MM activity for a wide array of clinically relevant drugs and drug classes in the context of BM microenvironment interactions and provides context for further validation and potential suitability for repurposing to treat MM within the medullary compartment. [Display omitted] DisclosuresAftab:Cleave Biosciences, Inc.: Research Funding; Omniox, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Onyx Pharmaceuticals, Inc.: Research Funding. Off Label Use: The use of tofacitinib citrate and ruxolitinib will be discussed in preclinical contexts for treatment of multiple myeloma. Other approved drugs and drug classes will be generally presented in similar off-label context..

Abstract 4202: Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia

Abstract Deletions and dominant-negative mutations in IKZF1, the gene encoding Ikaros transcription factor, are found in ∼85% of Ph+ B-ALL and in some cases of Ph− B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph− or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and ponatinib have been incorporated into chemotherapy regimens for Ph+ B-ALL, the majority of patients still relapse, which correlates with residual bone marrow disease following induction therapy. New therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph− B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) that mimics the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated that loss of Ikaros DNA-binding function arrests B-lymphoid development at a large pre-B cell stage that can give rise to B-ALL. Survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK downstream of integrins and growth factor receptors that plays important roles in cancer stem cell biology and the tumor microenvironment. Here, we show that dysregulated tyrosine kinases, including BCR-ABL1, cooperate with Ikaros mutation to accelerate the development of B-lymphoblastic leukemia in mice, correlated with a striking (∼30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Ikzf1-mutant BCR-ABL1+ lymphoblasts exhibit relative resistance to ABL1 TKIs including dasatinib that is dependent on the presence of stroma. VS-4718 is a potent, orally bioavailable FAK inhibitor that inhibits tumor growth and metastasis in preclinical models, and is currently under evaluation in clinical trials in patients with various solid tumors. VS-4718 treatment abolished stromal adhesion and induced apoptosis of Ikzf1-mutant B-ALL and synergized with dasatinib, but had little effect on Ikzf1 WT B-ALL cells. Primary human Ph+ B-ALL samples showed a correlation between IKZF1 mutation status, stromal adherence, and sensitivity to FAK and ABL inhibitors in vitro, and BM tropism in vivo in xenografted NSG mice. Results from ongoing in vivo therapy experiments will be presented. Together, these results suggest a new model for the pathogenesis of high-risk B-ALL and its resistance to conventional therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM leukemic stem cells. Inhibition of FAK signaling in Ph+ or Ph− IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. FAK inhibitors represent a promising new approach for the treatment of high-risk IKZF1-mutant B-ALL patients. Citation Format: Nilamani Jena, Ila Joshi, Toshimi Yoshida, Zhihong Zhang, Zhong-Ying Liu, Prasanthi Tata, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Katia Georgopoulos, Richard A. Van Etten. Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2015-4202

Abstract CT220: A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc

Abstract Background: Immunotherapy has recently emerged as a promising modality in cancer treatment, but little is known about the application of this modality in pancreatic cancer (PC). Tumor-infiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the PC cells in significant numbers due to the presence of stroma and a suppressive microenvironment. One of the leading causes for immune suppression is elevated expression of PD-L1 either by the tumor cells or the surrounding regulatory cells, resulting in dysfunction of TILs. Neoadjuvant chemoradiation therapy (CRT) has been advocated as a potential way to improve outcomes of patients with resectable or borderline resectable PC. More importantly, there is recent evidence to suggest that CRT can increase the presence of TILs in the PC microenvironment (PCME), leading to production of interferon-γ (IFN-γ), which could increase the expression of PD-L1 through a negative feedback loop. Accordingly, we hypothesize that blocking the PD-1 receptor will synergize with CRT to increase the density and activation of TILs in the PCME. Methods: This is a prospective multicenter randomized trial which will accrue subjects with resectable or borderline resectable pancreatic cancer who had not received prior treatment for PC. The primary objectives of the study are: (1) to determine the safety of neoadjuvant CRT in combination with Pembrolizumab. (2) To estimate the difference in the number of TILs in pancreatic cancer subjects receiving neoadjuvant CRT in combination with Pembrolizumab to the number of TILs in subjects receiving neoadjuvant CRT alone. This study will also investigate the effect of CRT+/-anti-PD-1 on the other effector and suppressive immune cells and immune checkpoints in PCME. Eligible subjects will be randomized 2:1 to the investigational treatment (Arm A) to receive Pembrolizumab administered IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine (825 mg/m2 orally twice daily, Monday through Friday, on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. In all subjects, restaging CT scan or MRI will be performed at 4-6 weeks after completion of neoadjuvant treatment to determine resectability. Patients without local or distant disease progression will be taken to the operative room for planned surgery (within 2 weeks of imaging). Postoperatively, resected patients will receive off study standard of care adjuvant gemcitabine (1000mg/kg IV weekly for 3 out of 4 weeks for 6 months). Post operatively resected patients will be followed for up for PFS and OS for up to 2 years. Citation Format: Matthew H.G Katz, Todd W. Bauer, Gauri Rajani Varadhachary, Reid B. Adams, Amy R. Lankford, Gina Petroni, Timothy N. Bullock, Craig L. Slingluff, Osama E. Rahma. A randomized multicenter phase Ib/II study to assess the safety and the immunological effect of chemoradiation therapy (CRT) in combination with Pembrolizumab (anti-PD1) to CRT alone in patients with resectable or borderline resectable pancreatic canc [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT220. doi:10.1158/1538-7445.AM2015-CT220

All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment.

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.

KRAS mutation in colorectal cancer and its association with a stromal-derived gene signature.

628 Background: KRAS mutation in colorectal cancer (CRC) is characterized by an altered transcriptional profile when compared to wild-type KRAS tumors. The list of differentially expressed genes overlaps significantly with a stromal fibroblast activation (SFA) signature present across multiple carcinomas. We have reported low expression of SFA genes in KRAS mutant CRC compared to KRAS wild type tumors. Here we sought to confirm the variation of the SFA signature with KRAS mutation and infer its origin in the stromal component of the tumor using experimental models. Methods: The SFA signature was assessed in an inducible-KRAS murine CRC model using RNA-sequencing, and in a CRC cell line with and without a transduced KRAS mutant vector by microarray analysis. Finally, RNA-sequencing of CRC patient-derived xenografts (PDXs) was used to determine whether the SFA signature was being expressed in the tumor epithelium or the surrounding stroma by leveraging the ability to align sequenced reads to the mouse and human genomes separately. Results: The SFA signature was identified in the inducible-KRAS mouse model, matching human cohort observations of decreased SFA gene expression in KRAS mutant CRC. On the other hand, KRAS transduction did not recapitulate the SFA signature in a CRC cell line, suggesting that the presence of stroma may be required for the expression of the SFA signature. Finally, RNA-seq reads for SFA signature genes in CRC PDXs immediately after implantation aligned primarily to the human genome but in later passages of the same PDXs aligned only to the mouse genome. These data suggest that the SFA transcriptional program is associated with the stroma rather than the epithelial tumor cells. Conclusions: KRAS mutation in CRC is associated with a gene expression signature derived from the tumor stroma. These findings suggest that KRAS mutation in the epithelial tumor cells may impact the tumor microenvironment in CRC.

A randomized multicenter Phase Ib/II study to assess the immunological effect of chemoradiation therapy (CRT) in combination with pembrolizumab compared to CRT alone in resectable or borderline resectable pancreatic cancer

Meeting abstracts Tumor-infiltrating lymphocytes (TILs) play a major role in anti-tumor immune responses, and their presence is correlated with survival in a variety of tumors. These TILs do not reach the pancreatic cancer (PC) cells in significant numbers due to the presence of stroma and a

Bone Marrow Stromal Cells Induce Bortezomib Resistance Partly through a Transcriptome Modulation in Multiple Myeloma Cells

INTRODUCTIONIt has been suggested that environment-mediated drug resistance (EMDR) might account for the intrinsic (de novo) resistance to therapy in some myeloma patients developing in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Understanding the mechanisms exploited by bone marroe stromal cells (BMSCs) to generate such resistance has long been sought. Furthermore, knowledge of the role that changes in expression of various genes could play is scanty. In this study we explored how BMSCs could induce resistance to bortezomib and whether it would change the expression of an array of genes in MM cells in the presence of bortezomib.METHODSUsing patient-derived BMSCs and a normal immortalized human BMSC line (HS-5), we set up a model in which several human myeloma cell lines (HMCLs), RPMI-8226, U266, MM1.S, OPM2 and H929, were co-cultured with stromal cells in the presence or absence of bortezomib. Drug cytotoxicity in HS-5 context was performed using XTT assay. Transwell system, Paraformaldehyde (PFA) fixation of BMSCs and treatment of BMSCs with Brefeldin-A (BFA, an inhibitor of intracellular protein transport) were used as controls to delineate the direct or indirect roles of BMSCs. Percentage of apoptotic cells was determined with anti-CD138/APC, annexin-V/FITC and PI staining in FACS. For gene expression array, co-cultures were applied to magnetic cell separation (EasySep, Stem Cell Technologies), total RNA was isolated from MM cell pellets and synthesized cDNAs were applied to real time PCR gene expression arrays. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in co-cultures compared to MM cell monocultures in the presence of bortezomib using the 2-ΔΔCtalgorithm.RESULTSIn the XTT cytotoxicity assay, all tested HMCLs displayed a higher level of viability in the presence of stroma than in stroma-free conditions (average bortezomib IC50s: stroma 22nM vs no stroma 8.53nM, p<0.02). Moreover, FACS analysis for all HMCLs showed that the percentage of bortezomib-induced apoptosis in stroma context was significantly lower than that in stroma-free conditions (p<0.05). Notably, in BFA and PFA treatment settings the level of bortezomib-induced apoptosis was lower (but not significant) than that in Transwell system suggesting that both soluble factors secreted by BMSCs and direct adhesion contribute to stroma-induced resistance to bortezomib. Gene expression analyses in real time PCR showed that the induced resistance was associated with downregulation of some genes involved in cell cycle and proliferation control (CDKN2A, CDC6, CDK2, EGR1, PTEN), downregulation of some genes involved in p53 signaling pathway (PUMA, P73, P53, MDM2, NOXA, BAX) and downregulation of some genes involved in apoptosis (CASP3, PARP).CONCLUSIONOur findings suggest that BMSCs induce resistance against cytotoxic and apoptotic effects of bortezomib in MM cells. To this end, in our tested HMCLs, the gene expression patterns may imply involvement of multiple pathways in controlling stroma-induced resistance to bortezomib. Although gene expression results were verified real time PCR readouts, changes in genes may not always correlate with protein changes. Hence, our study has given us the rationale to proceed with more in depth exploration as to whether BMSCs may render MM cells resistant to bortezomib by negative modulation of p53 signaling pathway controlling apoptosis and cell cycle. DisclosuresNo relevant conflicts of interest to declare.

Pacritinib Suppresses Leukemic Outgrowth from FLT3-ITD Positive Stroma-Adherent Primary AML Cells

Background: Targeting the stroma-AML cell interaction is a new dimension in anti-leukemia therapy. FLT3-ITD AML cells are known to derive protection from bone marrow stroma through up-regulation of resistance pathways such as MEK and JAK2. Pacritinib is a dual FLT3 and JAK2 inhibitor with equipotent activity for each target and is currently offered as stand-alone therapy for relapsed AML patients with FLT3-ITD mutant disease enrolled in the MRC AML 17 trial.Aim: We set up this study to determine if pacritinib as a single agent is sufficient to abrogate the stroma mediated resistance of AML, and to investigate leukemic signalling in stroma-adherent AML cells.Results: Pacritinib efficacy was assessed in 62 primary AML samples. FLT3-ITD samples were significantly more sensitive to pacritinib compared to FLT3 wild type AML samples after 48 hours exposure in vitro (mean IC50 92 nM vs 292 nM [range ITD 11-290 nM, range WT 19-2600 nm], p=0.01). Pacritinib induced apoptosis in AML cells but was not lethal to normal CD34+ bone marrow cells within the same dose range. FLT3-ITD AML cells cultured on MS5 stroma retained sensitivity to pacritinib after 7 days of co-culture (p<0.01, n=6).There was no significant effect on stromal cells. In this study, an MS5 stromal co-culture model was set up to enable interrogation of FLT3-ITD mutated primary AML cells that are adherent and non-adherent to stroma. PCR analyses performed on CD45+ adherent AML cells confirmed that they carried FLT3-ITD. Flow analyses demonstrated that the reduction of stroma-adherent AML cells was pacritinib dose dependent. Following 7 days of treatment and after drug wash-out, prior pacritinib exposure resulted in sustained suppression of leukemic outgrowth from stroma-adherent FLT3-ITD AML cells at 14 days of co-culture, compared to untreated cells (p<0.01, n=6). To establish if pacritinib could impact upon longer-term leukemic stromal adherence and survival in FLT3-ITD AML, we stained leukemic cells with Aldefluor and flow sorted the CD34+ 38- ALDH intermediate fraction, which has been shown to possess in vivo engrafting ability and to correlate with clinical relapse. Sorted cells were plated onto MS5 stroma and cobblestone area forming cell (CAFC) capacity determined as a function of pacritinib dose. Pacritinib reduced CAFC frequency at 5 weeks (from 500 nm, n=3).To investigate leukemic cell signalling, FLT3-ITD mutant AML cells were cultured on stroma in the presence of pacritinib for up to 24 hours, and intracellular flow analyses performed on adherent and non-adherent fractions. Control stroma-free cultures demonstrated significant p-STAT5 knockdown from 1 hour of drug exposure. In the presence of stroma, non-adherent AML cells did not show this reduction in p-STAT5. P-STAT5 down-regulation in adherent AML cells was delayed (to 18 hours), but exhibited dose dependency (p<0.05 at 500 nm pacritinib, p=0.0005 at 1 μM; n=6). Interrogation of JAK2 signalling showed up-regulation of p-JAK2 in adherent AML cells in the presence of pacritinib from 3 hours in 2 of 5 samples. There was no abrogation of AKT or ERK signalling in stroma-adherent or non-adherent fractions and this was confirmed by immuno-blotting. Interestingly, pacritinib was observed to decrease non-phosphorylated beta catenin levels in stroma-adherent AML cells and this was not seen in non-adherent AML cells (p=0.029, n=5).Short-term drug response Cell Glo luminescent assays reflected results from intracellular signalling experiments. Significant synergy was seen when pacritinib was combined with a MEK inhibitor (PD0325901) with accompanying down-regulation of ERK (mean CI 0.31 [range 0.19-0.44] at 5:1 ratio, n=20).Conclusion: These results demonstrate that pacritinib can suppress leukemic outgrowth from stroma adherent cells in FLT3-ITD driven AML in medium-term (14 days) and long-term (5 weeks) assays. However, short-term intracellular signalling profiles suggest that resistance pathways can remain active in some cases and may require AKT/ERK targeting for their eradication. Good synergy is seen with MEK inhibition, making this an attractive combination for clinical evaluation. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Studies of the IAP Antagonist Debio 1143 in Combination with Cytarabine or Doxorubicin in a Mouse Model of AML

Background: Relapse and drug resistance remains the major cause of therapy failure in Acute Myeloid Leukemia (AML). Inhibitors of apoptosis proteins (IAPs) are a family of proteins that suppress proapoptotic signaling and represent attractive targets to overcome chemotherapy resistance. Debio 1143 (a.k.a. AT-406) is an orally-active IAP antagonist able to promote apoptosis alone and/or in combination with chemotherapy. We recently reported the results of a Phase 1 study of Debio 1143 in combination with daunorubicin and cytarabine (araC) in poor-risk AML patients (DiPersio et al. J Clin Oncol 32:5s, 2014, a7029). Although Debio 1143 showed good tolerability and could be safely combined with chemotherapy, 62% (8/13) of patients with complete remissions relapsed within the study period. Here, we used a preclinical AML model to begin examining the effects of BM stroma on the efficacy of Debio 1143 combined with araC or doxorubicin (dox).Methods: We generated murine acute promyelocytic leukemia (APL) cell lines that are resistant to araC and dox both in vitro and in vivo by chronically exposing APL cells to increasing concentrations of drug in vitro. This APL model was previously generated by knocking in the human PML-RARa cDNA into the cathepsin G locus (Westervelt et al. Blood, 2003). We used WST-1 cell proliferation assays and bioluminescence imaging (BLI) to evaluate the efficacy of Debio 1143 to induce apoptosis alone and in combination with araC or dox in vitro in chemotherapy sensitive and resistant APL cells in the presence and absence of M2-10B4 stromal cells. Dox or araC was provided for (a) a 6h period initiated 24h before Debio 1143 (i.e., with an intervening 18h washout of the chemotherapy), (b) simultaneously with Debio 1143, or (c) 6h after Debio 1143. To assess Debio 1143 in vivo, we injected APL cells into syngeneic mice. In our first experiment, mice (n=5/cohort) were treated with PBS, araC (500 mg/kg, s.c.), dox (50 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 5, 6, 12, and 13 after APL injection. In our second experiment, mice (n=8/cohort) were treated with PBS, araC (200 mg/kg, s.c.), dox (10 mg/kg, s.c.), Debio 1143 (100 mg/kg, p.o.) or their combination on days 3 to 7 and 10 to 16 after APL injection. In both experiments, Debio 1143 was given 2 hours prior to chemotherapy.Results: Figure 1 shows that coculture of APL cells with stromal cells protects them from araC-induced killing (Figure 1A) and that our araC-resistant cells are indeed resistant to araC (Figure 1B). Treatment with Debio 1143 alone has no effect on parental or araC-resistant APL survival in the presence or absence of stroma cells. However, Debio 1143 in combination with araC killed the parental APL cells and overcame the stroma cell-mediated chemotherapy protection (Figure 1A). Although combination therapy partially inhibited araC-resistant APL cell proliferation in the absence of stroma, it had no effect when the cells were cultured in the presence of stroma (Figure 1B). We obtained similar results with dox and dox-resistant cells (data not shown). This in vitro data with the parental APL cell line was confirmed in vivo. Combination therapy with chemotherapy (araC or dox) and Debio 1143 for 4 days (EXP#1) or 10 days (EXP#2) significantly prolonged the overall survival of mice by 4 to 7 days compared to single agent therapy (Debio 1143, araC, or dox) in this aggressive tumor model (Table 1). Preliminary in vitro data suggests that pretreatment of parental or araC-resistant APL cells with chemotherapy (araC or dox) enhances their killing by Debio 1143 compared to simultaneous treatment.Conclusions: Debio 1143 combined with chemotherapy inhibits parental and chemotherapy-resistant APL growth in vitro and in vivo. Future mechanistic studies are required to determine how Debio 1143 can overcome stroma-mediated resistance. [Display omitted] [Display omitted] DisclosuresRettig:Debiopharm International SA: Research Funding. Vuagniaux:Debiopharm International SA: Employment. Zanna:Debiopharm International SA: Employment. DiPersio:Debiopharm International SA: Research Funding.

Abstract 4328: Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation

Abstract Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of replicationally quiescent mature B cells, as they fail to undergo apoptosis. Studies show that supporting stromal cells in the microenvironment provide survival advantage to CLL cells by conferring resistance to spontaneous and drug-induced apoptosis. A recent transcriptome characterization showed that compared to normal lymphocytes, genes involved in metabolic pathways were upregulated in CLL cells (Ferreira et al., Genome Res 2013). We hypothesized that stromal cells may have an active role on energy metabolism of malignant B-cells in order to cope with their energy demands. We assessed the major energy generating pathways (glycolysis and mitochondrial OXPHOS), in primary CLL cells using Extracellular Flux Analyzer (Seahorse Bioscience). The oxygen consumption rate (OCR) in CLL cells is surprisingly high compared to the proliferating lymphoma cell lines. Whereas, the glycolytic flux, measured as extracellular acidification rate (ECAR) is significantly low, suggesting that OXPHOS measured as OCR is upregulated in CLL cells; hence we focused on OXPHOS. Increased OCR was not impacted by age, Rai stage, lymphocyte counts, β2M, IgVH mutation status or LDH levels. However, ZAP70 positive (a poor-prognosis marker) samples had higher OCRs (n=30, p =0.002). Compared to males, samples obtained from female patients had higher OCRs (n=33, p = 0.043). Co-culturing with stroma (NK.tert human cell line) for 18-24 hrs had an impact on OCR in CLL; out of 30 samples, 24 showed a significant (p - 0.0005) increase in basal OCR and/or spare respiratory capacity (SRC, biological parameter of mitochondrial respiration). In addition, 11 of 12 CLL samples showed a significant increase in basal OCR, when plated on stroma without prior incubation (p &amp;lt; 0.0001), whereas the SRC did not statistically change (p - 0.9791). Moreover, the differences associated with gender and ZAP70 status were more pronounced when CLL cells were co-cultured on stroma (ZAP70 status, n= 30, p = 0.0013; Gender n=28, p =0.0173). In contrast to OCR, ECAR did not show stroma-induced statistical increase. Preliminary data with murine stroma line (M2-10B4) also showed upregulation of OCR in CLL cells. The stroma-mediated increase in OCR was not due to an increase in proliferation index; as CLL cells on stroma stained negative for Ki67 (marker for cell proliferation). Metabolite analysis by mass spectrometry of 5 CLL sample sets revealed that TCA cycle and gluconeogenesis were among the top 5 hits of pathways upregulated by stromal co-culture. At the molecular level, CLL cells in presence of stroma showed an increased p-AKT and p-ERK levels. Active AKT modulates GLUT4 protein levels, which was expressed in all 5 samples. Collectively, these data suggest that in CLL cells, OXPHOS (measured as OCR) is high and was further induced by stroma. Citation Format: Hima Venkata Vangapandu, Kumudha Balakrishnan, William G. Wierda, Michael J. Keating, Christine M. Stellrecht, Varsha Gandhi. Marrow stromal cells promote mitochondrial energy metabolism in primary CLL cells without impacting cellular proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2014-4328

Bone Marrow (BM) Stromal Expression Of Cytochrome P450 (CYP) Enzymes Protects Acute Myeloid Leukemia (AML) From All-Trans Retinoic Acid (atRA)

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved the outcome of acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of atRA resistance have been previously reported (i.e., over-expression of PRAME or up-regulation of Tal1 pathway), yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, although atRA induces complete remissions (CRs) in &gt;80% of cases, most patients eventually develop disease recurrence. We have recently showed that BM stromal control of RA signaling via expression of CYP26 is important in determining normal hematopoietic stem cell fate. Thus, here we hypothesize that the BM microenvironment is responsible for the difference between the in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. It is well-recognized that pharmacological concentrations of atRA (10-7M x 3 days) differentiates NB-4 cells [a t(15;17) APL line] as demonstrated by decreased cellular expansion (32.9±1 vs. 3.5±1.3, mean±SD of cellular expansion folds of control vs. atRA-treated cultures, n=3, p&lt;0.01) induction of cell cycle arrest (52.7% vs. 86.2% Go/G1, control vs. atRA), decreased clonogenic activity (61±12% of control in atRA cultures, n=3, p&lt;0.01), increased expression of differentiation markers (47.4±7% vs. 84.5±13% CD11b positive cells, control vs. atRA, n=3, p=0.01), disappearance of blasts (44.5±7% vs 24.3±4% blasts, control vs. atRA, n=4, p&lt;0.01) and emergence of morphological neutrophils. We observed similar pro-differentiation effects of atRA on other AML lines including HL-60 [non-t(15:17) APL line], KG-1 (primitive line arising from MDS), Kasumi-1 [t(8;21)] and OCI-AML3 (NPM1 mutated). When the incubations were repeated with the 5 AML lines in the presence of BM stroma, atRA activity (i.e., upregulation of differentiation markers, inhibition of clonogenic growth) was blocked. Inhibition of stromal CYP26 by the competitive inhibitor R115866, rescued the AML cell sensitivity to atRA: clonogenic recovery of NB4 cells was 92±17% on stroma with atRA compared to 56±15.9% when R115866 was added) (mean±SD of clonogenic activity of untreated stroma cultures, n=3, p=0.03) and 67.5±8.9% cells expressed the differentiation marker CD11b when treated with atRA on stroma compared to 95.1±4.9% with the combination of atRA+R115866 (n=3, p=0.03). Similar results were observed using HL-60, KG-1, Kasumi-1 and OCI-AML3 cells. CD34+ AML blasts from the BM of newly-diagnosed patients with t(8;21) AML were cultured as above. When isolated from the BM, the blasts showed only low-level expression of mature myelomonocytic markers (n=3). However, the expression of differentiation markers was significantly increased when cultured in serum (which contains about 10-9M atRA) and even further with the addition of 10-7M atRA (Figure). The presence of stroma during culture was associated with maintenance of an immature leukemic phenotype; moreover, as seen above, inhibition of CYP26 by R115866 restored AML sensitivity to atRA (Figure). CYP26 inhibitor had no effect on AML cells in stroma independent cultures. Our data suggest that CYP26 activity in the BM microenvironment creates retinoid low sanctuaries that protect AML cells from systemic atRA therapy. Thus, inhibition of CYP26 activity provides a new opportunity to expand the clinical activity of atRA in both APL and non-APL AML. It is also tempting to hypothesize that the P450-mediated detoxification of drugs by the stroma is not a retinoid-specific phenomenon but rather a more general, cell-extrinsic mechanism of drug resistance. Bone marrow stroma protects primary AML blasts from atRA induced differentiation Flow cytometry analysis of CD15 expression of CD34+ blasts isolated from the BM of a patient with t(8;21) AML. On day 0, these cells express no CD15. Culture in the presence of serum results in rapid acquisition of CD15 (liquid control). This is further enhanced by treatment with 10-7M atRA. Co-culture with BM stroma inhibits CD15 expression and atRA has only minimal effect in the presence of stroma. Inhibition of stromal CYP26 overcomes stroma’s inhibition of atRA-mediated up-regulation of CD15. Disclosures: No relevant conflicts of interest to declare.

Treatment With G-CSF Reduces Acute Myeloid Leukemia (AML) Blasts Viability In Presence Of Bone Marrow Stroma

Abstract Background The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC). Objective The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs. Methods and patients Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria. Results G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL). Conclusions G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients. Disclosures: No relevant conflicts of interest to declare.

PI3K-Alpha Plays A Major Role In Multiple Myeloma and Its Inhibition With BYL917 Decreases Proliferation, Synergizes With Other Therapies and Overcomes Stroma-Induced Resistance

IntroductionMultiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and ResultsWe analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. ConclusionThis is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures:Vali:Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.