HS-5 Conditioned Medium Research Articles

Mediator Kinase/CDK8 Inhibition As a Strategy to Improve FLT3 Inhibitor Activity in Acute Myeloid Leukemia

Acquired resistance to targeted FLT3 tyrosine kinase inhibitors (TKIs) remains a major barrier to durable clinical responses for patients with FLT3-mutant AML. Multiple resistance mechanisms to next generation FLT3 TKIs converge upon RAS/MAPK pathway activation, including protective cytokine stimuli of the bone marrow microenvironment or through secondary oncogenic RAS mutations. RAS mutations also decrease efficacy of the BCL2 inhibitor venetoclax now widely used in AML, suggesting an anti-apoptotic influence of MAPK signaling. These findings highlight a significant unmet need for novel therapeutic combinations to address MAPK-mediated resistance in AML. We report results from a genome-wide CRISPR interference (CRISPRi) apoptosis screen using FLT3-ITD mutant MOLM-14 cells treated with the FLT3 TKI gilteritinib while cultured in HS5 conditioned media (CM) to mimic cell-extrinsic MAPK-driven TKI resistance. We identified RNA pol II and Mediator kinase/CDK8 ascritical nodes for AML cell survival in the setting of FLT3 inhibition. We further demonstrated that CDK8 inhibition with the small-molecule SEL120 re-sensitized multiple FLT3-mutant AML cell lines and primary samples to gilteritinib. We also performed a second CRISPRi apoptosis screen to identify hits that sensitize to gilteritinib in a model of cell-intrinsic MAPK-mediated resistance using MOLM-14 cells that harbor secondary NRAS-G12C mutation.We used Enrichr to perform an integrated analysis and validated the Core Mediator Complex and CDK8 knockdown genes as cellular components significantly enriched among sensitizing hits in both screens. The Mediator subunit MED12 was identified as a top gene level hit that strongly sensitized cells to gilteritinib in both screens, further emphasizing that Mediator kinase and its transcriptional regulation may be a central pathway engaged in MAPK-dependent survival in FLT3-mutant AML. To better understand potential mechanisms of increased combinatorial CDK8i/FLT3i activity, we performed RNA-seq in MOLM-14 cells treated with gilteritinib, SEL120, or the combination in HS5 CM. Using GSEA we found that gilteritinib stimulated adaptive interferon/inflammatory gene signatures at 16h drug treatment, but this response was restrained by addition of SEL120. Additionally, we identified a striking upregulation of the lineage-controlling transcription factor IRF8 and its gene expression signature with concomitant silencing of SPI1 transcriptional output in cells treated with gilteritinib/SEL120. CDK8i treatment alone in AML has previously been shown to increase expression of super-enhancer (SE) associated genes, including IRF8. Interestingly, SPI1was also a significant gene-level hit in both CRISPRi screens. Using CRISPRi we repressed IRF8 expression in MOLM-14, MOLM-14 NRAS-G12C, and MV411 (another FLT3-mutant line) cells and found this significantly blunted the combinatorial pro-apoptotic effect of SEL120, implying that induction of IRF8 expression plays a significant role in driving response to combined gilteritinib/SEL120. We finally assessed in vivo activity of gilteritinib combined with SEL120. We first performed pilot toxicity studies in NSG mice and determined that gilteritinib 30 mg/kg, SEL120 30 mg/kg, or the combination dosed 5 days/week by oral gavage (OG) were regimens well tolerated by mice. We then engrafted luciferase-tagged MOLM-14 and MOLM-14 NRAS-G12C cells into NSG mice and treated for 4-weeks with gilteritinib, SEL120, or the combination. We found gilteritinib/SEL120 extended survival in both MOLM-14 and MOLM-14 NRAS-G12C models. Similar efficacy studies are ongoing in FLT3-mutant patient-derived xenograft (PDX) models with NSGS mice and will be reported. Our results indicate that FLT3-mutant AML cells employ an adaptive inflammatory response to evade FLT3i-induced apoptosis, and CDK8i represses this adaptation while concomitantly increasing the ratio of IRF8 to SPI1-driven gene expression programs. We speculate this latter effect may in part alter an AML cell's differentiation state to sensitize cells to apoptosis, though further study to explore this hypothesis is needed. Our in vitro and in vivo efficacy data further validated combined FLT3i/CDK8i as a promising investigational strategy to pre-empt or overcome MAPK-mediated FLT3 TKI resistance.

Navtemadlin, a Novel MDM2 Inhibitor, Potentiated Venetoclax-Induced Antitumor Efficacy in TP53 Wild-Type Acute Myeloid Leukemia (AML)

Introduction: The tumor suppressor p53 ( TP53) maintains genomic integrity, and nearly 50% of cancers are characterized by a loss or mutation in the TP53 gene. Murine double minute 2 (MDM2) negatively regulates p53 expression and promotes oncogenesis in TP53 wild-type ( TP53 WT) tumors. Navtemadlin (nvtm) is a potent, selective, orally available MDM2 inhibitor that overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis in TP53 WT tumors. AML is a heterogeneous hematological malignancy resulting from the transformation of stem cells and is characterized by poor therapy response. Thus, combination therapies are required to avoid tumor escape mechanisms, such as alterations in apoptotic machinery and metabolism. Venetoclax (ven), a BCL-2 inhibitor, is a promising therapy in AML. Overexpression of anti-apoptotic members of the BCL-2 family, including myeloid cell leukemia-1 (MCL-1) limits ven response, thus requiring MCL-1 inhibitors to further improve sensitivity. A proof-of-concept study with idasanutlin (idasa), a first-generation MDM2 inhibitor and ven demonstrated manageable safety and encouraging efficacy in relapsed/refractory AML patients (Daver et al. Blood 2023). Here, we present a preclinical evaluation of nvtm alone and in combination with ven in the setting of TP53 WT AML, with a focus on metabolic pathways, pro-survival signaling, and bone marrow (BM) niche. Methods: AML-derived cell line, MOLM-13 ( TP53 WT, FLT3-ITD) was cultured alone, with HS5 AML stromal cells or with cytokines. Cultures were treated with DMSO, nvtm (0.1-1.5 μM), or ven (0.5 μM) for short-term (4-8 hrs) or long-term (48-72 hrs) and analyzed for changes in cell cycle progression, viability, clonogenicity potential, and metabolism (glycolysis and mitochondrial oxidative phosphorylation by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively). Primary AML BM mononuclear cells (MNCs) were cultured in cytokine support media. Results: In MOLM-13 cells, short-termnvtm (0.5 µM) treatment rapidly restored p53 expression and phosphorylation, with greater efficacy than idasa. Long-term treatment significantly induced apoptosis in a dose-dependent manner from 0.25 μM (p<0.01) with 80% cytotoxicity at 0.75 μM (p<0.001). Cell cycle analysis at 48 hr showed that nvtm significantly arrested cells in the sub-G0-G1 phase. Clonogenicity potential was significantly lowered in nvtm-treated cells. Metabolic alterations in the Mito Stress and Glycostress tests showed nvtm significantly lowered EACR, OCR, and mitochondrial membrane potential, as compared to idasa. We next evaluated the effects of nvtm in ven-treated MOLM-13 cells. Combination treatment significantly lowered the metabolism, cell viability, and clonogenicity potential of MOLM-13 cells, compared to nvtm or ven treatment. Immunoblotting analysis revealed a reduction in key anti-apoptosis proteins (BCL-2, MCL-1, PARP). Strikingly, nvtm downregulated ven-induced MCL-1 expression in a dose-dependent manner. Culturing MOLM-13 cells in HS5-conditioned media, transwells overlying HS5 cells, or with HS5 cells increased MOLM-13 cell resistance to nvtm. However, in HS5 co-cultures, a combination of nvtm and ven strongly re-sensitized MOLM-13 cells for apoptosis. Nvtm also significantly blocked the cytoprotective effects of AML-relevant cytokines (IL-6, SCF, FLT3 ligand, TPO, and GM-SCF). In cultures of primary AML BM-MNCs (n=11), nvtm alone at clinically relevant concentrations reduced viability (14-40%), and a combination nvtm and ven significantly enhanced AML cytotoxicity (30-52%). Conclusions: Nvtm, a potent second-generation MDM2 inhibitor, rapidly restored p53 function, induced apoptosis in p53 WT AML MOLM-13 by impeding cell cycle, viability, clonogenicity potential, and reducing both glycolysis and oxidative phosphorylation. Nvtm combined with ven significantly enhanced these effects. Importantly, this combination blocked stromal cell-mediated (contact and soluble factors) cytoprotection. These encouraging results provide a mechanistic basis for the evaluation of this combination in vivo and as a therapeutic strategy to overcome documented ven resistance mechanisms in TP53 WT AML patients.

Abstract 2825: Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines

Abstract Multiple myeloma (MM) is a disease of malignant plasma cells that resides in the bone marrow microenvironment (BMM). While advances in therapy have improved patient outcomes, the majority of patients relapse. Clinical trials have shown that BCMA targeted CAR-T therapy induces MM remission in most patients with heavily pretreated relapsed refractory MM; however, the duration of the response has been disappointing. CAR-T cells induce target cell death through two primary mechanisms, granzyme B and FASL. To define the role of FAS-induced cell death in CAR-T therapy in MM, we first evaluated if FAS-induced cell death was important for CAR-T activity in MM. We utilized CRISPR-Cas9 to generate FAS KOs in three MM cell lines: RPMI8226, KMS18, and OCIMY5. These lines were utilized in vitro in cytotoxic CAR-T assays where MM cells were cultured with BMCA CAR-T cells at five effector to target (E:T) ratios (range: 0:1 to 1:1). The loss of FAS protected KMS18 and OCIMY5 from CAR-T induced cell death, changing the E:T ratio needed to induce 50% MM cell death 3.2 and 3.4 fold respectively, but did not protect RPMI8226. We determined the expression of FAS negatively correlated with the EC50 of rFASL, suggesting that FAS expression levels are important for this response. To determine the role of the intrinsic apoptotic pathway in FASL-induced death, we knocked out BAK and BAX in KMS12 PE and OCIMY5. This did not protect KMS12 PE or OCIMY5 from rFASL-induced cell death, suggesting that FAS uses the type I mitochondria-independent pathway in these cell lines. The BMM supports MM progression and promotes drug resistance in part by altering the apoptotic threshold. Therefore, we examined the impact of the BMM on FASL-induced cell death. Coculture of MM cells with the HS5 stromal cell line protected KSM12 PE and KMS18 from rFASL, with the EC50 of rFASL changing 2.72 and 3.1 fold respectively, but did not protect OCIMY5. To determine if the protection observed was cell contact dependent, or only required soluble factors, we utilized HS5 conditioned media (CM). HS5 CM protected KMS12 PE, KMS18, and surprisingly OCIMY5 from rFASL, causing a change in EC50 from control of 1.65 - 2.23 fold. CM lacking IL6 did not protect MM cells from rFASL. Therefore, we tested the effects of IL6 addition IL6(10 ng/ml) and observed protection from rFASL in KMS12 PE and KMS18 cells with a change in EC50 of 2.03 and 3.84 fold respectively; however, we did not see protection in OCIMY5. To identify the mechanism of IL6-induced resistance to rFASL, we cultured BAK/BAX KO cells in HS5 CM and observed protection of these apoptosis-deficient KMS12 PE and OCIMY5 cells from rFASL. This suggests that soluble factors in the BMM can convert cells from type I to type II (mitochondrial-dependent) death receptor signaling and that inhibiting BMM signals such as IL6 may enhance FASL-induced cell death in MM. However, the data also demonstrate that the role of FAS in CAR-T killing is cell context dependent. Citation Format: James C. Ackley, Samuel McCachren, Sagar Lonial, Damien J. Green, Stanley R. Riddell, Geoffrey R. Hill, Madhav V. Dhodapkar, Lawrence H. Boise. Stromal cell derived IL6 inhibits the extrinsic apoptotic pathway in multiple myeloma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2825.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML.Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS.Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio.Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. DisclosuresLogan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Stroma-Based Versus Stroma-Free Long-Term Survival and Proliferation of Primary Acute Myeloid Leukemia (AML) Cells

Background: Acute myeloid leukemia (AML) is a very aggressive hematological malignancy that carries an overall poor prognosis. High throughput screening of primary (patient-derived) leukemic cells is becoming increasingly valuable as a preclinical tool to help individualize the treatment of relapsed/refractory AML patients. However, the necessity of robust in-vitro culture conditions has often limited the optimization of high throughput drug sensitivity testing. Our lab has previously validated a high throughput assay in 384-well format that utilizes primary leukemic cells maintained in a cocktail of cytokines tested for chemosensitivity and resistance against anti-leukemic agents (Shum et al. Clinical Lymphoma, Myeloma and Leukemia 2013). However, cytokines have not been shown to support long-term proliferation of primary AML cells; nor does it assess drug candidates within the appropriate tumor microenvironment. Stromal cells of the bone marrow microenvironment, however, have been reported to support not only the growth and maintenance of normal hematopoiesis, but also the long-term, in-vitro growth of leukemic cells, while also influencing their response to chemotherapy agents. A comprehensive comparison of primary AML cell growth under our validated cytokine assay and stromal conditions has not previously been undertaken. In this study, we compared the long-term proliferation and phenotypes of primary AML cells in high-throughput format under different stromal conditions against cytokines only. Results from this study would help optimize parameters for a high-throughput drug sensitivity testing comparing the differential response to anti-leukemic agents under cytokine versus stromal conditions.Methods: The proliferation and immunophenotypes of relapsed/refractory primary AML peripheral blood samples were assessed in 96-well plates under four culture conditions: 1) cell culture medium only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned medium or 4) in a cocktail of four cytokines as per our previously validated cytokine-based assay. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 (stromal marker) were determined by flow cytometry weekly for 2 weeks.Results: Our results demonstrated a very heterogeneous response in the growth of primary AML cells among the different patient samples to support with cytokines, HS-5 conditioned medium, and direct contact with HS-5 stromal cells. Overall, primary AML cells continued to expand after 2 weeks in both cytokine and stromal support. The majority of primary AML samples demonstrated higher proliferation with direct contact with stromal cells compared to cytokines only. AML cells growth with cytokines or HS-5 conditioned medium was also comparable. Our initial investigation also revealed one patient sample with a CD34+/CD38+ predominant phenotype, which demonstrated a > 600-fold expansion within 2 weeks with cytokines only - greater than 3-times the expansion seen by direct contact with HS-5 stromal cells. Immunophenotypes were maintained for all culture conditions at 2 weeks.Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that cytokine support not only maintained, but can also continue to expand primary AML cells after 2 weeks, independent of stromal support. Consistent with previous studies, we also confirmed that HS-5 stromal cells support long-term in-vitro expansion of primary AML cells, which cannot be substituted by HS-5 conditioned medium. As expected, the majority of primary AML cells showed higher proliferation with direct contact with HS-5 stromal cells compared to cytokine support, but based on one of our patient's sample, which showed a significantly higher expansion with cytokine support only, it is possible that there is a subset of patients whose cell growth respond better to cytokines. This study represents the first direct comparison between the effects of cytokines and two different stromal conditions on the long-term growth of primary AML cells in high throughput format, while providing support that these in-vitro culture conditions can be independently utilized to compare the differential primary AML cell response to anti-leukemic agents. DisclosuresFrattini:Lin Bioscience: Consultancy; Astellas: Honoraria.

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Marrow Stromal Cells Secrete FGF2 in Exosomes, Providing Paracrine Protection for Leukemia Cells from Kinase Inhibitors

Direct Comparison of in-Vitro Growth of Primary Human Acute Myeloid Leukemia (AML) Cells in Stromal and Stromal-Free Conditions

Background: Acute myeloid leukemia (AML) is a very aggressive bone marrow malignancy which carries a poor prognosis despite intensive chemotherapy. The treatment of relapsed and refractory AML remains suboptimal. Although more novel therapies are being introduced for AML, there is a limitation of appropriate, predictive, preclinical models available to identify and test novel therapies. In-vitro drug sensitivity testing of patient-derived AML cells is increasingly being used to facilitate treatment options. However, these tests are often done in suboptimal conditions with difficulty in the interpretation of results. Accumulating evidence has shown that the long-term, in-vitro, survival of primary AML cells can be supported with stromal co-culture, which would also take into account the influence of the surrounding tumor microenvironment. However, there has been no direct comparison of a stromal co-culture method to non-stromal growth method of primary AML cells. This is the first comprehensive direct comparison of the proliferation and immunophenotypes of primary AML cells under two different stromal conditions and cytokines, the results of which would not only highlight the importance of stromal cells to chemotherapy resistance but also lay the groundwork for the feasibility and importance of comparing it to non-stromal drug sensitivity testing.Methods: The proliferation and immunophenotypes of seven bio-banked, relapsed/refractory primary AML peripheral blood samples were assessed in 6-well plates under four culture conditions: 1) cell culture media only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned media or 4) in a cocktail of four cytokines. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 were determined by flow cytometry weekly for 2 weeks.Results: Our results demonstrated a heterogeneous growth response among the seven patient samples in response to the four growth conditions. However, the end effect was that cytokines, HS-5 conditioned media and HS-5 stromal cells can all support the long-term proliferation and viability of the majority of primary AML samples. These three conditions showed statistically significant higher growth compared to the same primary cells cultured in media alone (p-values < 0.05). The degree of expansion was still significantly higher for those cells with cytokines than those with HS-5 conditioned media or stromal cells. Six of the 7 patient samples expanded with cytokine, up to 4-fold by 2 weeks of culture. In comparison 5 of the 7 patient samples expanded up to 3-fold with HS-5 stromal cells, and 4 of the 7 patient samples also expanded up to 3-fold with HS-5 conditioned media at 2 weeks of culture. For the 4 patients, whose cells were supported by both stromal cells and conditioned media, the degree of expansion was similar. Unexpectedly, one out of 7 patient samples had significantly higher expansion compared to all of the other patients in all growth condition, with a 9-fold expansion in cytokine and conditioned media, and a 12-fold expansion in HS-5 stromal cells. There was also a 3-fold expansion in media alone. Baseline AML immunophenotypes were preserved after 2 weeks of culture for all 4 growth conditions, with minimal differentiation.Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that each experimental condition alone can expand primary AML cells. In addition, cytokine support alone can expand primary AML cells at 2 weeks of culture for the majority of patient samples, independent of stromal support, a finding that has previously not been reported. Consistent with previous studies, our data also supports the conclusion that stromal support maintains long-term in-vitro expansion of primary AML cells. All three experimental conditions showed an overall effect of growth with high viability and maintenance of immunophenotypes at 2 weeks of culture. The direct comparison of drug screening under these three experimental conditions would permit the exploration of the protective effect of the bone marrow microenvironment on AML cells allowing for the identification of individualized therapeutic options, while also allowing for comparison to non-stromal based in-vitro assays. DisclosuresNo relevant conflicts of interest to declare.

Combining Dasatinib and AC220 Reduces Stroma-Based pSTAT5Y694 in FLT3-ITD+ AML and Overcomes FLT3 TKI Resistance

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220.We found that FLT3-ITD+ cell lines and primary cells grown in HS-5 CM exhibited strong upregulation of pSTAT3Y705 and pSTAT5Y694 that was unaffected by treatment with 10nM AC220. In RM, pSTAT3Y705 was undetectable irrespective of AC220 dose and pSTAT5Y694 was abolished by 10nM AC220. shRNA knockdown experiments of STAT3 and STAT5 in FLT3-mutated cell lines grown in CM and treated with AC220 showed that STAT5 knockdown alone consistently resulted in greater inhibition of cell proliferation than STAT3 knockdown alone. This suggests that in FLT3-ITD+ AML cells cultured in CM, upregulation of pSTAT5Y694 by stroma-derived soluble factors is the main contributor to AC220 resistance.We decided to use a candidate approach to identify the upstream kinases leading to pSTAT5Y694, focusing on inhibitors of pathways previously implicated in STAT5 activation in AML. We used phosphoflow to quantify pSTAT5Y694 in MOLM-13 cells grown for 24 hours in CM in the presence of 10nM AC220 combined with ruxolitinib, dasatinib, ibrutinib, PD173074 (FGFR1i) or PRT062607 (SYKi). Results from this screen indicated that the combination of dasatinib and AC220 most effectively decreased pSTAT5Y694 in MOLM-13 cells cultured in CM (minimum dasatinib dose =100nM). To further validate dasatinib as a candidate for combination drug treatment, we performed cell proliferation experiments in MOLM-13 cells cultured in CM for 72h with graded concentrations of AC220 and the candidate inhibitors at a minimum candidate drug dose of 100nM. Of the four inhibitors tested, the combination of dasatinib and AC220 most significantly decreased the IC50 of AC220 in MOLM-13 cells grown in CM (IC50 AC220 alone: 11.09nM; IC50 AC220 + 100nM dasatinib: 0.41nM; p<0.05, n=3).It has recently been reported that FLT3-ITD AML cells harbor a highly glycolytic phenotype that can be partially suppressed by FLT3 TKIs (Huntly et al, Blood, 2018). Our preliminary Seahorse experiments show that the combination of dasatinib and AC220 more effectively inhibits glycolysis in MOLM-13 cells than AC220 alone, with differential modulation in CM versus RM. These results suggest that dasatinib inhibits a metabolic pathway critical to maintaining glycolysis in MOLM-13 cells challenged by AC220-based FLT3 inhibition. Moreover, these data indicate that extrinsic signals contribute to metabolic reprogramming of FLT3+ AML cells, enabling survival despite FLT3 inhibition. Experiments are underway to delineate how CM contributes to metabolic resistance and how dasatinib intercepts this survival signal. Our results support the concept of combining AC220 with dasatinib to reduce residual leukemia in FLT3+ AML and prolong survival. Further investigation is ongoing and will be reported at the time of abstract presentation. DisclosuresDeininger:Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Preclinical Activity of Novel MCL1 Inhibitor AZD5991 in Multiple Myeloma

Epigenetic Drug Combination Chemo-Sensitizes Pediatric AML By Reducing Cell Adhesion and Dislodging AML Cells from the Bone Marrow

BackgroundWe showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML.MethodsAML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee.ResultsAML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B).We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C).To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy.ConclusionOur data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. [Display omitted] DisclosuresKolb:Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.

Stroma-Based Activation of pSTAT3Y705 Confers Resistance to FLT3 Inhibitors in FLT3 ITD-Positive AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm that carries the worst prognosis among the hematologic malignancies. Up to 30% of AML patients exhibit activating mutations in FLT3 tyrosine kinase. FLT3 internal tandem duplications (ITDs) comprise ~70% of these mutations and are associated with a poor prognosis. Most patients treated with a single-agent FLT3 tyrosine kinase inhibitor (TKI) relapse within months due to secondary mutations in the FLT3 tyrosine kinase domain (TKD). Results from trials of FLT3 TKIs in AML reveal that leukemic blasts are more easily cleared from peripheral blood than from bone marrow (BM), suggesting that the BM microenvironment promotes survival of AML cells, including leukemia initiating cells, despite inhibition of FLT3. In this conceptual framework, extrinsic factors allow AML cells to survive TKI exposure until AML cell-intrinsic resistance is conferred by FLT3 TKD mutations, leading to clinical relapse. Here, we investigated the role of the BM microenvironment in protection of FLT3+AML cells from treatment with AC220 (quizartinib), a clinically available FLT3 TKI.To investigate the potential of the BM microenvironment to mediate TKI resistance in AML, we cultured FLT3-ITD+ AML cell lines, including MOLM-13, MOLM-14 and MV411, and the CML cell line, K562 (control; FLT3 wild-type), with graded concentrations of AC220 under the following conditions: (i) in regular medium (RM), (ii) in direct contact (DC) with human HS-5 BM stromal cells, or (iii) in HS-5 conditioned medium (CM). Cell proliferation and apoptosis assays revealed that, in RM,AC220 reduced proliferation and increased apoptosis of MOLM-13, MOLM-14 and MV411 cells, but had no effect on K562 cells. DC greatly reduced the effects of AC220 in all three FLT3-ITD+ AML cell lines, with comparable results observed between DC and CM. To confirm these data using primary cells, CD34+ blasts from a patient with newly diagnosed FLT3-ITD+ AML were similarly cultured in RM versus CM ± AC220. Consistent with results in cell lines, CM rescued primary AML cells from AC220-mediated cell death. These data indicate that soluble factors from the BM environment protect FLT3-ITD+ cells from the effects of FLT3 inhibition.Our lab and others have demonstrated that HS-5 DC and CM activate STAT3 in chronic myeloid leukemia, which mediates resistance to BCR-ABL1 TKIs (Bewry et al. Mol Cancer Ther 2008, Traer et al. Leukemia 2012, Eiring et al. Leukemia 2015). To interrogate the role of STAT3 in BM-mediated protection of AML cells from FLT3 inhibition, all cell lines were assessed for pSTAT3Y705 and total STAT3 by immunoblot analysis under each culture condition. In FLT3-ITD+ AML cells grown in RM, pSTAT3Y705 was undetectable, irrespective of AC220 dose. In contrast, pSTAT5Y694 was readily detected at steady state and suppressed by AC220. AML cells cultured in HS-5 DC or in HS-5 CM exhibited strong upregulation of pSTAT3Y705 that was unaffected by AC220, suggesting that soluble factor(s) promote STAT3 activation in AML. pSTAT5Y694, on the other hand, was slightly elevated by HS-5 DC or CM, but remained under control of FLT3 kinase activity. In order to mechanistically implicate STAT3 activation in stroma-based protection, we used a retroviral shRNA construct to knockdown STAT3 (shSTAT3) compared to an empty vector control (LMP) in MOLM-14 cells. STAT3 knockdown (~70%) was confirmed by qRT-PCR and immunoblot analyses. Cells containing shSTAT3 and LMP were cultured for 72 hours in RM or CM ± AC220, followed by analysis using MTS assays. As expected, CM increased the IC50 of AC220 from 1.37 nM to 6.24 nM in LMP-expressing cells (n=3). In contrast, shSTAT3 reduced the IC50 of AC220 from 6.24 nM to 2.87 nM (n=3) in CM, with minimal effects in RM. Similarly, pharmacologic inhibition of STAT3 using the novel STAT3 inhibitor, BP-5-087 (Eiring et al. Leukemia 2015), reduced the IC50 of AC220 from 10.07 nM to 5.91 nM in CM. Analogous experiments in additional FLT3-ITD+cell lines and primary AML cells, using shSTAT3, dominant-negative STAT3 constructs and BP5-087 are ongoing.Our data suggest that STAT3 is a critical signaling node in FLT3-independent TKI resistance mediated by the BM microenvironment. Therapeutic strategies designed to combine FLT3 and STAT3 inhibition may inhibit the survival of leukemic cells in the BM niche, thereby preventing subsequent clinical relapse conferred by TKD mutations. DisclosuresDeininger:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Targeting of JAK/STAT Signaling to Reverse Stroma-Induced Cytoprotection Against BCL2 Antagonist Venetoclax in Acute Myeloid Leukemia

Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib.To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial.Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs.To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1).In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.

Bone Marrow Stroma Protects BCR-ABL-Negative Myeloproliferative Neoplasms from Ruxolitinib- and Vorinostat-Induced Apoptosis

Background: The classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are characterized by increased proliferation of hematopoietic precursors in the bone marrow resulting in elevated numbers of terminally differentiated cells. Despite the recent advances in the understanding of the biology of MPN, there is still no curative treatment for MPN except for bone marrow transplantation. The discovery of JAK-STAT constitutive activation in the majority of MPN patients led to the development of clinical studies targeting MPN with Ruxolitinib, a JAK1/2-specific inhibitor. However, despite achieving significant reductions in splenomegaly and symptomatic improvement in MPN patients, JAK inhibition failed to induce complete remissions and eradicate the malignant clone. In alternative, histone deacetylase inhibitors have shown success in the treatment of several hematological malignancies but their efficacy in MPN is limited.Aims: Ruxolitinib and the HDAC inhibitor Vorinostat both fail to eradicate the neoplastic clone in MPN patients. Here, we tested the hypothesis that the bone marrow stroma confers resistance to these drugs by preventing their cytotoxic effects on neoplastic cells.Material and Methods: MPN patient derived cell lines (SET-2 and HEL) were cultured alone, with HS-5 bone marrow stroma or with HS-5 conditioned medium in the presence of clinically-relevant concentrations of Ruxolitinib and Vorinostat. At different time points, cellular viability was analyzed by staining with Annexin-V/PI and CD45-APC (to distinguish MPN cells from the HS-5 cells). Furthermore, cells were lysed and RNA was extracted for gene expression analysis. Activation of specific signaling pathways was determined by Western-blot using phospho-specific antibodies for effectors of the following signaling pathways: PI3K-Akt-mTOR; MEK-Erk; MAPK-JNK; NF-κB and JAK-STAT.Results: Treatment of SET-2 and HEL cells with both Vorinostat and Ruxolitinib promoted apoptosis and decreased proliferation. Importantly, apoptosis was significantly abrogated when MPN cells were cultured in the presence of a stromal layer of HS-5 cells or HS-5 conditioned medium. The stroma protective effect was maintained for up to 6 six days and was also dependent on the concentration of the drugs. These effects correlated with altered expression in MPN cells of genes associated with inflammatory processes, apoptosis and proliferation (CDKN1A, IER3, BIRC3, TNFRSF8, TNFRSF9, COX2, IL1B, XIAP), and with the activation of signaling pathways important for cellular homeostasis, such as PI3K-Akt-mTOR, MAPK-JNK, JAK-STAT and NF-κB as shown by increased phosphorylation of Akt (Ser473); GSK3α/β (Ser9/Ser21); S6 (Ser235/236); STAT3/5 (Tyr705/Tyr694) and IKBα (Ser32).Summary/Conclusions: Overal, we show that bone marrow stroma protects MPN cells from the cytotoxic effects of two clinically effective pharmacological agents of different classes: Vorinostat and Ruxolitinib. This protective effect is likely achieved, through the up-regulation of genes that inhibit apoptosis (BIRC3, TNFRSF8, TNFRSF9) and also through the activation of pro-survival signaling pathways such as PI3K-Akt-mTOR. We did not observe any effects of the bone marrow stroma on proliferation or differentiation, suggesting that the main effect of stromal cells is to prevent apoptosis of the neoplastic cells. Our results identify a possible cell non-autonomous mechanism by which Ruxolitinib fails to eradicate the neoplastic clone in MPN patients. Through the identification of the factors released by the bone marrow stroma, we hope to uncover why the bone marrow protects MPN cells and possibly define novel therapeutic strategies maximizing the use of Ruxolitinib and Vorinostat in MPN. DisclosuresAlmeida:Shire: Speakers Bureau; Amgen: Speakers Bureau; Novartis: Consultancy; Celgene: Consultancy; Bristol-Meyer Squibb: Speakers Bureau.

Direct Contact With Bone Marrow Stromal Cells Protects CML Progenitors From Imatinib Through Cytoplasmic Stabilization Of β-Catenin

A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by >3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses.CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013).To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations. Disclosures:Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Interleukin-6 Enhances the Survival of Myeloma Cells by Regulating Bim Binding to Anti-Apoptotic Bcl-2 Proteins

AbstractAbstract 4024The survival of multiple myeloma cells in the bone marrow is largely dependent on its interactions with cellular components of the microenvironment. These interactions, in the form of cell-cell interactions and soluble factors support myeloma cell survival and allow for the mutations associated with transformation to drive plasma cell growth and proliferation. Moreover survival signals from the bone marrow microenvironment influence the response to therapeutic agents, therefore understanding the molecular basis of these interactions is necessary to improve the depth of responses to therapy in myeloma patients.Cell survival is regulated by the Bcl-2 family of proteins and we previously reported that in myeloma cells one can map which Bcl-2 protein is necessary for cell survival by determining which protein is sequestering the pro-apoptotic molecule, Bim. This could also be used to predict myeloma cell sensitivity to th Bcl-2/Bcl-xL antagonist ABT-737. While studying the response of freshly isolated patient samples to ABT-737, we found that in 40% of the samples tested (N=10) as well as in 5 myeloma cell lines, inclusion of stromal components significantly inhibited ABT-737-induced apoptosis. Thus the microenvironment can influence survival by altering the pattern of Bcl-2 dependence in cells. We have determined that a soluble factor secreted by stromal cells is necessary for this activity and now report that this factor is interleukin-6.We found that addition of conditioned medium from the HS-5 stromal line or freshly isolated bone marrow stromal cells (N=4) from myeloma patients or normal donors (N=1) to human myeloma cell lines all resulted significant resistance to ABT-737. Since all of these cells are a rich source of IL-6, we tested if the addition of recombinant IL-6 mimicked the addition of stromal cell supernatant. Indeed rIL-6 (10 ng/ml) was as potent as co-culture with HS-5 cells or addition of HS-5 or stromal cell conditioned medium at blocking ABT-737-induced apoptosis in MM.1s cells. Thus IL-6 is sufficient to block ABT-737-induced apoptosis. To demonstrate that IL-6 was necessary we determined the effect of the addition of an IL-6 neutralizing antibody on stromal cell-induced resistance to ABT-737. We found that addition of the antibody to HS-5 conditioned medium blocked resistance to ABT-737 in a dose-dependent fashion. Moreover we found that addition of anti-IL6 to a patient sample had a similar effect as removing the stromal cells on the response to ABT-737-induced death. Taken together these data suggest that IL-6 is both necessary and sufficient for regulating Bcl-2 dependence in multiple myeloma.To define the molecular basis for these findings we initially investigated the expression of the key Bcl-2 proteins involved in Bcl-2 dependence and myeloma cell survival. Despite their profound effects on ABT-737-induced apoptosis we found that neither co-culture of MM.1s cells with HS-5 cells, HS-5 conditioned medium or rIL-6 had any effect on the expression of Mcl-1, Bcl-xL or Bim. We then investigated the patterns of interaction between these proteins and found that the addition of stromal components or IL-6 resulted in a shift of Bim binding from Bcl-xL to Mcl-1. This is consistent with our previous findings that when Bim is bound to Mcl-1, myeloma cells are less sensitive to ABT-737. Finally we set out to determine how IL-6 signaling results in a change in ABT-737 sensitivity. We treated MM.1s cells with ABT-737 and IL-6 in the presence of inhibitors of the MEK/ERK, p38, PI3K/AKT, and JAK/STAT pathways. We found that only the MEK inhibitor, U0126 could re-sensitize IL-6-treated cells to ABT-737-induced death at concentrations that did not kill cells or have additive effects with ABT-737 in the absence of IL-6 (P<0.05 2-way ANOVA). Taken together, these data indicate that stromal cells regulate myeloma cell survival through IL-6 by altering Bim binding in a MEK-dependent fashion. This results in a cell that is highly dependent on Mcl-1. These data offer an explanation for why proteasome inhibitors are so effective in this disease. While the bone marrow microenvironment is enforcing Mcl-1 dependence, proteasome inhibitors induce the Mcl-1 inhibitor Noxa, which overcomes this activity of IL-6. Moreover these data point to potential ways of combining the targeting of Bcl-2 proteins and IL-6 signaling as a means to effectively kill myeloma cells in the bone marrow microenvironment. Disclosures:Kaufman:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx Pharmaceuticals: Consultancy.

STAT3 Inhibition Synergizes with BCR-ABL1 Inhibition to Overcome Kinase-Independent TKI Resistance in Chronic Myeloid Leukemia (CML)

Abstract 31In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity.To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p<0.008) and increased apoptosis (17.9% increase, p<0.03) of parental K562 cells following imatinib exposure, thereby abolishing the protective effect of extrinsic BM-derived factors. In contrast, shSTAT3 had no effect on cells grown in regular medium. In intrinsic resistance, shSTAT3 reduced clonogenicity of K562R cells by 58.3% (p<0.0004) and AR230R cells by 62.2% (p<0.002) in the presence of 1.0 μM imatinib, with no effect on TKI-sensitive controls. A dnSTAT3 mutant lacking the C-terminal activation domain also impaired the clonogenicity of K562R and AR230R cells by 36.1% (p<0.003) and 50.0% (p<0.004), respectively. Thus, STAT3 plays a functional role in mediating intrinsic and extrinsic TKI resistance in BCR-ABL1-expressing cell lines.Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p<0.03) and AR230R cells by 72.5% (p<0.0004), with no effect on TKI-sensitive controls. When CMLCD34+ progenitor cells from newly diagnosed patients (n=4) were cultured in the presence of HS-5 CM and treated with the SF1-066/imatinib combination, clonogenicity following drug exposure was reduced by 45.2% (p<0.002) compared to imatinib. Therefore, pharmacologic inhibition of STAT3 and BCR-ABL1 overcomes intrinsic and extrinsic TKI resistance in both CML cell lines and CMLCD34+ progenitor cells.We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p<3.6×10−6) and 51.8% (p<8.6×10−6), respectively, with little effect on cells expressing a scrambled reporter. Selected inhibitors then provided scaffolds for the synthesis of focused libraries with enhanced potency and selectivity. Immunofluorescence data revealed that two of these inhibitors, BP5-087 and BP5-088, resulted in cytoplasmic accumulation of pSTAT3Y705 in CMLCD34+ progenitors grown in HS-5 CM, consistent with a block of dimerization. Screening and validation of third-generation STAT3 inhibitors are underway and will be reported.Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures:No relevant conflicts of interest to declare.

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.

Bone Marrow Stromal Cells Enforce MCL-1 Dependence In Multiple Myeloma Through the Secretion of A Soluble Factor,

Abstract 3950It is well established that the bone marrow microenvironment contributes to the growth and survival of multiple myeloma plasma cells, however the molecular mechanisms that control these changes within the myeloma cell have not been completely elucidated. We have recently demonstrated that myeloma cell lines can be dependent on the anti-apoptotic protein MCL-1 or can be co-dependent on MCL-1 and Bcl-2/xL. The primary distinction between MCL-1 dependence and co-dependence is the distribution of the pro-apoptotic BH3-only protein Bim. In MCL-1-dependent lines, Bim is primarily associated with MCL-1. In contrast in co-dependent lines Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it can not bind to MCL-1 because of the presence of the MCL-1 inhibitor, Noxa. This renders these cells sensitive to the Bcl-2/xL inhibitor ABT-737. We have confirmed these findings in freshly isolated myeloma cells and demonstrated that among the 4 patient samples tested, Bim was associated with both MCL-1 and Bcl-xL and the cells were sensitive to ABT-737. This suggests that co-dependence on MCL-1 and Bcl-xL may be a common phenomena in myeloma. We now demonstrate that in about 50% of patient samples tested, CD138 column-purified MM cells are more sensitive to ABT-737 than cells that were not purified following ficoll separation of the bone marrow aspirate. This suggests that stromal components in the marrow alter the dependence on Bcl-2 proteins resulting in a cell that is more MCL-1 dependent when in the presence of stroma. To directly test this possibility we determined the effect of co-culturing the co-dependent line, MM.1s with the human stromal line Hs-5 on ABT-737 (0.6 mM) sensitivity and Bim binding. MM1.s cells were incubated with Hs-5 cells for 30 min prior to addition of drug or were cultured in medium containing 50% conditioned medium (48 h growth) from Hs-5 cells to determine if any effects observed were due to a soluble factor. Following a 24 h culture, cells were subjected to CD138 column purification and apoptosis analyzed by Annexin V-FITC/PI flow cytometry. While mono-cultured MM1.s were very sensitive to ABT-737 (88.5% +/− 3.6% control apoptosis, mean +/− SE), the addition of Hs-5 cells or conditioned medium had a profound effect on cell survival resulting in significant resistance to Bcl-2/xL inhibition (23% +/− 4.4% and 24.5% +/− 2.3% control apoptosis respectively). To determine the molecular basis in this shift from co-dependence to MCL-1 dependence, western blot and co-immunoprecipitation analyses were performed. While neither co-culture nor conditioned medium had any significant effect on the expression of levels of Bim, MCL-1 or Bcl-xL, both conditions resulted in a shift of Bim binding from being equally split amongst MCL-1 and Bcl-xL to a binding pattern where Bim was preferentially bound to MCL-1. This is consistent with loss of sensitivity to ABT-737 and a shift to MCL-1 dependence. We next tested the effects of Hs5 co-culture and conditioned medium on other inducers of cell death. We focused on bortezomib (5 nM) and arsenic trioxide (2 mM) as both can induce the MCL-1 inhibitor Noxa, therefore sensitivity would not be expected to be significantly influenced by a change from co-dependence to MCL-1 dependence. Consistent with this possibility we found that bortezomib induced cell death was only modestly affected by co-culture (79% +/− 2.3% vs. 66.5% +/− 1.9% control apoptosis) and no protection was observed with Hs5 conditioned medium. Similar results were observed with arsenic trioxide (monoculture – 31% +/− 2.3%, Hs5 co-culture −15% +/− 1.0%, conditioned medium – 30% +/− 4.2% control apoptosis). Taken together these data demonstrate that a soluble factor produced by stromal support can alter Bim binding to anti-apoptotic Bcl-2 family members, rendering myeloma cells primarily dependent on MCL-1 and protecting them from cell death signals that function through inhibition of Bcl-2/xL. Moreover these data in part explain why bortezomib can overcome survival signaling provided by stromal support, as proteasome inhibition is capable of inducing the MCL-1 inhibitor Noxa. Disclosures:Kaufman:Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Boise:University of Chicago: Patents & Royalties.

Intrinsic and Extrinsic Survival Signals Converge on STAT3 As a Critical Mediator of BCR-ABL-Independent Tyrosine Kinase Inhibitor Resistance,

Abstract 3742In Philadelphia chromosome positive leukemia, mutations in the BCR-ABL kinase domain are a well established mechanism of resistance to tyrosine kinase inhibitors (TKIs), but fail to explain many cases of primary and acquired resistance. Extrinsic survival signals from the bone marrow microenvironment can protect CML cells from the effects of TKIs in a STAT3-dependent manner. To define the role of pSTAT3 in extrinsic versus intrinsic TKI resistance, we used BCR-ABL+ cell lines exhibiting ‘extrinsic resistance’ through culture in conditioned media (CM) from human HS-5 bone marrow stromal cells (Re), or derivative lines expressing native BCR-ABL with ‘intrinsic resistance’ adapted for growth in the presence of 1.0–2.5 μM imatinib (Ri).Immunoblot analysis showed near-complete suppression of BCR-ABL tyrosine kinase activity upon exposure to imatinib in all Re and Ri cells tested, suggesting BCR-ABL-independent resistance in both cases. K562-Ri and AR230-Ri cells proliferating in 1.0 μM imatinib showed upregulation of pAKTS473, pJAK2Y1007/1008, pSTAT3, and pSTAT5 compared to sensitive cells under the same conditions. When imatinib was increased (2.5 μM), pJAK2Y1007/1008 and pSTAT5 were markedly reduced, suggesting regulation by BCR-ABL kinase activity. In contrast, levels of pSTAT3 and pAKTS473 were further increased. Similar to Ri cells, extrinsically resistant cells (K562-Re, LAMA84-Re, KBM5-Re) showed increased pSTAT3 when cultured in HS-5 CM; however, upregulation was not observed for pAKTS473. Of key importance, pSTAT3 levels were also increased by HS-5 CM in CD34+ cells from newly diagnosed CML patients.To further investigate the role of pSTAT3 in TKI resistance, we used lentiviral shRNA to knockdown STAT3 (shSTAT3) in sensitive and resistant BCR-ABL+ cells. Cells were kept in culture for 24 hours with and without 1.0 μM imatinib in regular medium or CM, followed by trypan blue exclusion and/or plating in semisolid medium. Compared to cells expressing a scrambled control (shSCR), shSTAT3 reduced the in vitro growth of intrinsically resistant K562-Ri and AR230-Ri cells by 54.6% and 33.3% in the presence of imatinib (1.0 μM), respectively. Consistent with these observations, shSTAT3 impaired the clonogenic potential of K562-Ri cells by 65.0% following culture in 1.0 μM imatinib, and similar results were obtained for AR230-Ri cells, indicating that STAT3 plays a functional role in mediating intrinsic TKI resistance in multiple BCR-ABL-expressing cell lines. In extrinsic resistance, 3–12 hours of culture with CM from HS-5 stromal cells enhanced the clonogenic potential of K562-Re and LAMA84-Re cells by 25–40% in the presence of imatinib compared to controls cultured in regular medium. However, the protective effects of CM were abrogated by introduction of shSTAT3. In contrast, shSTAT3 had no effect on colony formation following growth in regular medium, consistent with the low level of pSTAT3 in parental K562 and LAMA-84 cells. These data suggest that STAT3 activation is a prominent feature of both extrinsic and intrinsic BCR-ABL-independent imatinib resistance. Additionally, preliminary experiments implicate pSTAT3 in intrinsic BCR-ABL-independent resistance to the third-generation TKI, ponatinib (AP24534), suggesting that activated STAT3 may have a role in resistance to other TKIs.Altogether, our data suggest that intrinsic and extrinsic pathways converge on STAT3 as a critical mediator of BCR-ABL-independent TKI resistance, and implicate this pathway as a potential therapeutic target for the treatment of patients with TKI resistance despite BCR-ABL inactivation. We posit that microenvironmental cues activate pathways that initially support survival of leukemia cells despite BCR-ABL inhibition, and that overt resistance develops when these cells establish an intrinsic mechanism to maintain activation of the same pathways. Disclosures:No relevant conflicts of interest to declare.

Leukemia Microenvironment-Specific Galectin-3 Expression of Leukemic Cells Promotes Malignant Niche Formation and Bone Marrow Lodgment of Leukemic Cells in Chronic Myelogenous Leukemia

Abstract 1678We recently identifid that galectin-3 (Gal-3) is specifically induced in leukemic cells due to the support of the bone marrow microenvironment and promotes cell proliferation and resistance in leukemic cells to a variety of drugs, including imatinib mesylate, dasatinib, or genotoxic agents, especially in the case of chronic myelogenous leukemia (CML). In the current study, we continued and extended our study in order to identify the role of Gal-3 in the disease development of CML. First, we used a transmigration assay to investigate the role of Gal-3 in cell migration of leukemic cells. HS-5 conditioned medium (CM/HS-5) was used as the source of bone marrow stromal cells (BMSCs)-derived chemotactic stimuli. HS-5 is an immortalized human BMSC-derived cell line, which potently secretes G-CSF, GM-CSF, M-CSF, Kit ligand, MIP-1a, and interleukin (IL)-6, IL-8, or IL-11. When MYL cells, a CML cell line, were compared with Gal-3 overexpressing MYL cells (MYL/G3), which were generated by means of gene introduction, the latter showed a greater capacity for cell migration induced by CM/HS-5. Next, we investigated the involvement of Gal-3 in malignant niche formation, since recent studies have hypothesized that leukemic cells excrete growth factors which stimulate, via paracrine and autocrine loops, the growth of adjacent leukemic cells as well as of bone marrow supporting cells such as BMSCs or endothelial cells. When MYL cells and MYL/G3 cells were cultured with media containing conditioning medium (CM) from MYL cells and MYL/G3 cells in various ratios (designated as CM/MYL and CM/MYL/G3, respectively), both MYL cells and HS-5 cells proliferated more at higher concentrations of CM/MYL/G3, indicating that MYL/G3 cells excrete more growth factors for the MYL cells themselves as well as BMSCs. These findings were the same for cases with CML cell line K562 and Gal-3 overexpressing K562. We finally examined the in vivo role of Gal-3 in CML. Approval was obtained from the institutional review board at Kyoto University Hospital for the use of mice for this study which was conducted in accordance with the ethical principles of the Declaration of Helsinki. Fourteen male NOD/SCID mice at 6 weeks of age were sublethally irradiated (2 Gy) and 1.0×106 MYL cells (Group A) or 1.0×106 MYL/G3 cells (Group B) were transplanted intravenously via their tail veins into seven mice each. Body weight (BW) and the percentage of leukemic cells in peripheral blood (PB) were monitored at least twice a week. Although transplanted leukemic cells increased in a similar manner in the PB of both groups during the first three weeks, the number of PB leukemic cells of Group A mice then gradually decreased, while those of Group B mice remained the same until death. Although we had initially hypothesized that Group B might have a shorter survival than Group A, the actual result was the opposite, with the survival of Group A being significantly shorter than that of Group B (p =0.025). Surprisingly, the sites of disease involvement at death showed major differences between the two groups. Most mice from Group A showed extensive extramedullary involvement, such as intra-abdominal, mediastinal and/or subcutaneous tumors isolated from BM, while only one of seven mice showed BM involvement at the time of death. In contrast, all mice in Group B showed BM involvement, which sometimes outgrew BM, but none showed tumors isolated from BM. These results indicate that Gal-3 overexpression may facilitate BM homing and lodgment of CML cells. We also speculate that the reason for the shorter survival of Group A is that the tumors expanded much faster once leukemic cells had advanced outside BM and that this may have had a significantly more deleterious effect on the mice in Group A than on those in Group B. In conclusion, the findings of our study suggest that BMME-induced Gal-3 in leukemic cells plays a crucial role in disease development of CML, especially in the BM milieu. Disclosures:No relevant conflicts of interest to declare.