scholarly journals Targeting E-Selectin with GMI-1271 Overcomes Microenvironment-Mediated Resistance to Venetoclax/HMA Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Kyung Hee Chang ◽  
Muharrem Muftuoglu ◽  
Weiguo Zhang ◽  
Mahesh Basyal ◽  
Lauren B Ostermann ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Pdx Model ◽  
Bm Niche ◽  
Combinatorial Treatment

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (Ven/HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Despite improved remission rates, the duration of response is still inadequate. Adhesion to the bone marrow (BM) niche is critical for AML initiation, progression and leukemic stem cell (LSC) survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) is responsible for the tethering and rolling of leukocytes on perivascular endothelial BM niche cells (EC). In leukemia, E-sel has crucial roles in BM homing and engraftment (Krause et al, 2006). Through patient-derived AML xenograft (PDX) models and single cell proteomics, we have elucidated the roles of E-sel in AML survival and drug resistance. A PDX model derived from a patient who had developed resistance to Ven/HMA were treated with the E-sel antagonist, GMI-1271 (uproleselan; GlycoMimetic, Inc). We found that targeting E-sel mobilized human AML cells and sensitized them to Ven/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with Ven/HMA in comparison to Ven/HMA alone (p < 0.05). The synergistic effects of the combinatorial treatment on AML-PDX mouse survival were determined by Kaplan-Meier analysis. The combination of GMI-1271 and Ven/HMA significantly prolonged the survival of mice compared to vehicle control (p = 0.015) as well as the Ven/HMA (p = 0.0009) and GMI-1271 groups (p = 0.03). The median survival of the vehicle control, GMI-1271, Ven/HMA, and combination-treated groups of mice was 86, 91, 81.5, and 106.5 days, respectively. Histological analysis of BM, spleen, lung and liver demonstrated differences in leukemia cell infiltration, confirming enhanced anti-leukemia efficacy of the combination treatment. To delineate the mechanism of E-sel at the onset of drug mediated changes in AML signaling signatures, we employed another PDX model (Flt3-ITD and WT1 mutations, sorafenib-resistant). PDX mice with advanced AML (more than 20% human AML cells circulation in peripheral blood) were administered Ven/HMA, GMI-1271, or combination for 2 days. Single cell proteomics analysis by CyTOF determined that combinatorial treatment diminished levels of Ki67, IDU, and pRb compared to vehicle control or Ven/HMA alone, resulting in decreased proliferation of AML blasts. Activation of eNOS to produce nitric oxide (NO) through PI3K/AKT kinase, maintains clonogenic cell growth in malignant cells. A recent publication has demonstrated that introduction of NOS blockers in combination with chemotherapy led to slower leukemia progression and longer remissions in contrast to chemotherapy alone (Passaro et al, 2017). Interestingly, we observed reduced activation of PI3K and AKT in AML blasts as well as in BM CD31+EC cells in the GMI-1271 treated PDX model. eNOS phosphorylation was subsequently decreased in EC, suggesting that inhibition of E-sel may protect BM vasculature by blocking the production of NO. In addition, targeting E-sel showed signaling alterations in AML MSC. Administration of E-sel antagonist increased mTOR expression in MSC from AML-PDX. Combination treatment induced higher Ki67 positivity, and hyperactivation of pRb and p-S6 in MSC in vivo. Our group and others have recently reported that Ven-resistant AML cells exhibit an increased dependence on alternate anti-apoptotic proteins, Mcl-1 and Bcl-xl (Konopleva et al., 2016). We found that concomitant treatment in vivo with GMI-1271 and Ven/HMA further decreased the expression of Bcl-xl and Mcl-1 in AML blasts compared to Ven/HMA alone, suggesting a critical role for E-sel antagonists in overcoming drug resistance. E-selectin binding potential and focal adhesion kinase activity in AML blasts were decreased upon acute administration of pharmacological E-sel inhibitor. Other oncogenic signaling pathways including MAPK, p-S6, and STAT3, were all inhibited by the addition of GMI-1271 to Ven/HMA. Collectively, our results provide first evidence that an E-sel targeting strategy with GMI-1271 can overcome microenvironmental resistance to Ven/HMA-based therapy in AML by cancer cell autonomous and non-cell autonomous mechanisms in the BM vascular niche. Figure Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Weiguo Zhang ◽  
Kyung Hee Chang ◽  
Mahesh Basyal ◽  
Yannan Jia ◽  
Lauren B Ostermann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Underlying Mechanism ◽  
Leukemia Cells ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Pdx Model ◽  
Selectin Ligands

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 891-891
Author(s):  
Annamaria Gulla ◽  
Eugenio Morelli ◽  
Mehmet K. Samur ◽  
Cirino Botta ◽  
Megan Johnstone ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Clinical Outcome ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Escape ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Hong Mu-Mosley ◽  
Lauren B Ostermann ◽  
Ran Zhao ◽  
Challice L. Bonifant ◽  
Stephen Gottschalk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Perturbation of Genomic Instability by Wortmannin in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1105-1105
Author(s):  
Jagannath Pal ◽  
Dheeraj Pelluru ◽  
Mariateresa Fulciniti ◽  
Samir B Amin ◽  
Leutz Buon ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tumor Growth ◽  
Board Of Directors ◽  
Gene Product ◽  
Genomic Instability ◽  
Research Funding ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Over Time

Abstract Abstract 1105 Poster Board I-127 Genetic recombination plays a critical role in telomere maintenance, chromosomal translocation, and gene amplification, and may therefore underlie the chromosomal aberrations observed with high frequency in number of malignancies. The molecular mechanism/s inducing genomic instability remains ill-defined and their elucidation may provide methods to prevent tumor progression and development of drug resistance. Our earlier work has demonstrated that homologous recombination (HR) activity is elevated in multiple myeloma (MM) cells and leads to increased rate of mutation and progressive accumulation of genetic variation over time. We have further demonstrated that the inhibition of HR activity in MM cells by siRNAs targeting recombinase leads to significant reduction in the acquisition of new genetic changes in the genome; and conversely, induction of HR activity leads to significant elevation in the number of new mutations over time, and development of drug resistance in MM cells. Here we have evaluated a PI3K inhibitor Wortmaninin which has significant inhibitory activity against both HR and non-HR (nHR) pathways. Exposure of MM cells (OPM1, ARP and RPMI 8226) to wortmannin (WM) led to reduced expression of recombinase (hsRAD51) and nearly complete inhibition of HR activity, within 24 hrs as determined by a plasmid based assay in which generation of active gene product by recombination is measured. Similarly nHR was evaluated by measuring generation of intact gene product from a linearized plasmid. We evaluated effect of WM on nHR by 3 hours preincubation before transfecting the plasmid followed by cell culture for 72 hrs at 37° C. Cells were harvested and analyzed for nHR as previously described. Treatment with WM led to >40% reduction in nHR, indicating that WM affects both HR and NHR pathways. Downregulation of these pathways by wortmannin was also associated with a reduced growth rate of myeloma cells in culture by 20-25% at 48 hours. Importantly, WM treatment markedly decreased the acquisition of new genomic changes in MM as measured by genome-wide loss of heterozygosity assay as an indicator of genomic stability. To evaluate the impact of WM on in vivo tumor growth, OPM1 cells were injected subcutaneously in SCID mice and following appearance of palpable tumors, mice were treated with WM at 0.75 mg/kg, injecting daily intraperitoneally. Treatment with WM was associated with almost complete inhibition of tumor growth in vivo. Long term exposure of myeloma cells to WM was consistently associated with reduced telomere length, probably by blocking HR dependent ALT pathway. These data identifies dysregulated recombination activity as a key mediator of DNA instability and progression of MM, and WM as a potential therapeutic agent for prevention of myeloma progression and possibly drug resistance. Disclosures Anderson: Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3204-3204
Author(s):  
Alessandro Natoni ◽  
Mariah Farrell ◽  
Heather Fairfield ◽  
Lucy Kirkham-McCarthy ◽  
Matt Macauley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Sialyl Lewis X ◽  
Advisory Committees ◽  
Selectin Ligands

Abstract Introduction Multiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect. Methods E-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells. Results Treatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin. Conclusions Sialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. Disclosures O'Dwyer: Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Overcoming Drug Resistance in Myeloma By Synchronized Delivery of Therapeutic and Bone Marrow Disrupting Agents By Nanoparticles Targeting Tumor-Associated Endothelium

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1931-1931
Author(s):  
Cinzia Federico ◽  
Barbara Muz ◽  
Jennifer Sun ◽  
Kinan Alhallak ◽  
Justin King ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Tumor Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Dual Drug

Abstract Proteasome inhibitors (PIs) have improved the treatment of multiple myeloma (MM) and prolonged patient survival, but several challenges remain to overcome drug-resistance and toxicity. Bone marrow microenvironment (BMM) drives tumor progression and PIs-resistance in MM; and agents that inhibit the interaction between MM and BMM have been shown to re-sensitize MM cells to therapy. However, the synchronized in vivo delivery of BMM-targeting agents with PIs has been a challenge so far. Nanoparticles offer a valuable platform to encapsulate drugs, and if functionalized, they can facilitate specific delivery to tumor, thus improving treatment efficacy and reducing off-target effects. Within the BMM, the endothelium plays a relevant tumor promoting role. By analyzing the expression of an array of markers in normal and in MM-related endothelium, we found high levels of P-selectin expression on MM-activated endothelial cells (ECs) than normal cells and on ECs collected from the BM of either MM patients or MM-bearing mice compared to their respectively healthy BMMNCs. We next sought to develop lipid nanoparticles (LNPs) targeting the MM-related endothelium, loaded with both PI and BMM-targeting agent for synchronized delivery and reversal of the BMM-induced drug resistance. At this aim, we developed targeted LNPs towards P-selectin by decorating their surface with P-selectin-glycoprotein-ligand-1 (PSGL-1). PSGL-1-targeted LNPs showed specific binding to recombinant P-selectin than identically non-targeted particles, and to MM-associated endothelium compared to healthy endothelium, both in vitro and in vivo. To reverse BMM-induced resistance, LNPs were loaded with bortezomib (BTZ) together with a BMM disrupting agent, ROCK-inhibitor (Y-27632) that inhibits the downstream signaling of the RhoA GTPase pathway, known to be instrumental to the interaction of MM cells with BMM. Consequently, we tested the effect of synchronized delivery of BTZ and Y-27632 in the same LNP on MM cell survival in co-culture with the BMM in vitro. While Y-27632-loaded LNPs did not affect cell proliferation, LNPs loaded with both Y-27632 and BTZ enhanced responsiveness of MM cells to BTZ, compared to BTZ-loaded LNPs, thus overcoming the BMM-induced resistance. Mechanistically, we observed more significant inhibition of PI3K and MAPK signaling, decrease of pRb and up-regulation of p21 and induction of pro-apoptotic pathway (caspase-3, caspase-9 and PARP) by drug-loaded LNPs, compared to free drugs. In addition, drug-loaded LNPs were able to decrease adhesion and impair the migration of MM cells to ECs. We also investigated the in vivo efficacy of BTZ/Y-27632-loaded PSGL-1-targeted LNPs in a humanized murine model of MM. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls. The synchronized delivery of both agents using dual drug-loaded PSGL-1-targeted LNPs delayed the MM tumor progression and prolonged survival significantly more than all the controls (vehicle, BTZ and Y-27632 alone or in combination as free drugs, or encapsulated in non-targeted or in PSGL-1-targeted LNPs) demonstrating that both P-selectin targeting and combination of Y-27632 with BTZ reverses the BMM-induced drug resistance and enhances the efficacy of therapy in vivo. Altogether, our data demonstrate the ability of PSGL-1-decorated LNPs to specifically target MM-BMM; to efficiently encapsulate and deliver drugs to tumor tissue; to overcome BMM-induced drug resistance in vitro and in vivo, to reduce tumor growth and prolong overall survival. This study provides the preclinical basis for future clinical trials using MM-BMM-targeted nanomedicine able to enhance the effect of PIs or other drugs for the treatment of MM. Disclosures Roccaro: GILEAD: Research Funding; AMGEN: Other: Advisory Board. Vij:Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Azab:Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding.

scholarly journals The Transmembrane Receptor Roundabout 1 (ROBO1) Is Necessary for Multiple Myeloma Proliferation and Homing to the Bone Marrow Niche

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 507-507
Author(s):  
Giada Bianchi ◽  
Peter G. Czarnecki ◽  
Matthew Ho ◽  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Tumor Volume ◽  
Plasma Cells ◽  
P Value ◽  
Advisory Committees ◽  
Bm Niche ◽  
C Terminus ◽  
Necessary And Sufficient

Introduction Multiple myeloma (MM) is characterized by dissemination and accumulation of plasma cells in the bone marrow (BM), which promotes tumor cell growth and therapy resistance. ROBO1 is a conserved transmembrane receptor of the Ig superfamily with no intrinsic catalytic activity, and its role in MM pathogenesis is unknown. Material and Methods We first analyzed ROBO1 expression via western blot and/or immunohistochemistry (IHC). Gene expression profiling in a cohort of 170 newly diagnosed MM patients (IFM170) was used to compare ROBO1 expression across primary MM and BM stroma cells (BMSC), and normal BM plasma cells (PC). We used short hairpin RNA (shRNA) for stable ROBO1 knock down (KD) and CRISPR-Cas9 for ROBO1 knock out (KO). For protein structure-function and rescue studies, ROBO1 KO MM cells were transduced with a lentiviral vector expressing either full-length (FL) or truncated ROBO1 mutants devoid of extracellular (Cyt) or intracellular domain (DeltaCyt), including patient-derived truncating mutations, with a C-terminus triple FLAG tag. FLAG immunoprecipitation (IP) followed by mass spectrometry or western blotting and immunofluorescence (IF) were used to identify ROBO1 interacting partners and ROBO1 cellular localization. We used a hydrogel encapsulation system to study proliferation in a 3D system. To study extramedullary and intramedullary MM growth in vivo, WT and ROBO1 KO OPM2 were injected either subcutaneously (plasmacytoma model) or intra-medullary in femoral bones of donor mice which were then implanted subcutaneously in recipient SCID mice (µ-SCID model). PET-CT was used to assess tumor volume. Mouse tumors were retrieved for IHC and RNA extraction followed by RNA sequencing. To study dissemination and homing, KO and FL addback OPM2 cells were injected intravenously in SCID mice. Femurs and plasmacytoma were retrieved at endpoint for IHC. Results ROBO1 is highly expressed in human MM cell lines and primary MM cells with highest expression in cells carrying the high risk t(4;14) cytogenetic and low/absent expression in normal PC. Of human cancer cell lines, ROBO1 expression was limited to late B cell lineage; and ROBO1 KD was selectively cytotoxic against MM, but not other hematologic cancers. ROBO1 KO significantly decreases proliferation in a 3D culture system and tumor growth in extramedullary (mean tumor volume KO versus WT plasmacytoma: 457 versus 1323 mm3, p value= 0.02) and intramedullary (mean tumor volume KO versus WT: 823 versus 2684 mm3, p value= 0.001) murine models of human MM. ROBO1 KO MM cells show decreased adhesion to BM endothelial and BMSC, which is fully rescued by FL ROBO1 addback. To address whether ROBO1 loss alters dissemination/homing of MM cells in vivo, we injected mice intravenously with ROBO KO or FL addback OPM2 cells. While ROBO1 KO resulted in a modest, non-statistically significant prolongation in mouse OS (90 versus 75 days, respectively, p value 0.2), the pattern of disease was strikingly different. As expected, ROBO1 FL mice developed hindlimb paralysis with extensive BM infiltration with MM. Importantly, ROBO1 KO mice demonstrated reduced BM infiltration and developed solitary plasmacytoma. We next showed that ROBO1 C-terminus domain is necessary and sufficient to rescue ROBO1 KO proliferative defect while expression of ROBO1 truncations, including patient-derived frameshift mutations, acted as dominant negative. IP showed avid interaction of ROBO1 with ABL1. Interestingly, we showed that the cytosolic domain of ROBO1 undergoes cleavage and translocates to the nucleus, where its function is now being studied. Conclusions We show that ROBO1 is necessary for MM homing to the BM niche and for MM growth within and outside the BM space. ROBO1 cytosolic domain undergoes proteolytic cleavage and translocates to the nucleus and is necessary and sufficient to rescue ROBO1 KO defective proliferation. Based on our data, we propose a dual model for ROBO1 in MM: the full transmembrane receptor is involved in regulating adhesion, dissemination and homing of MM cells within the BM niche; the cleaved intracellular C-terminus domain participates in transcriptional regulation, promoting MM proliferation. These data suggest that ROBO1 C-terminus may be a novel molecular target in MM. Disclosures Roccaro: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding; AstraZeneca: Research Funding; Transcan2-ERANET: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding. Ghobrial:Takeda: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

scholarly journals Combination of Venetoclax and Ibrutinib Increases bcl2-Dependent Apoptotic Priming, Reduces ITK-Phosphorylation and Is Clinically Promising in Relapsed/Refractory T-Prolymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3965-3965
Author(s):  
Christoph Kornauth ◽  
Charles Herbaux ◽  
Bernd Boidol ◽  
Chantal Guillemette ◽  
Patrick Caron ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Single Agent ◽  
Substantial Reduction ◽  
Advisory Committees ◽  
Prolymphocytic Leukemia ◽  
Clinical Responses ◽  
Combinatorial Treatment

Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy with poor response to current treatment strategies. We recently demonstrated single agent activity of venetoclax in relapsed/refractory (r/r) T-PLL, however resistance developed on mono-therapy. We here set out to identify partners for an effective combinatorial treatment concept. Methods To overcome bcl-2 inhibitor resistance we utilized primary T-PLL patient samples and applied a combinatorial next-generation functional drug screen for venetoclax and 25 additional therapeutic agents (Fig.1a). Molecular mechanisms of drug combinations were evaluated by BH3-family member profiling and mass spectrometry. Protein expression was assessed by Western Blot and viability by AnnexinV/Hoechst staining. The best scoring combination was evaluated in two late stage r/r T-PLL patients. Results Pairwise combinations screen of venetoclax with candidate small molecule inhibitors and chemotherapeutic drugs on primary T-PLL cells revealed synergistic action of venetoclax with ibrutinib, idelalisib, and 5-azacytidine, and to lower extents Olaparib, Temsirolimus, Ruxolitinib and Belinostat whereas cisplatin antagonized the effect of venetoclax across all patient samples tested (Fig 1b). The combination of venetoclax and ibrutinib resulted in substantial reduction of viability in primary T-PLL cells (Fig 1c). BH3-profiling on primary T-PLL samples with and without ibrutinib treatment demonstrated enhanced overall priming with predominant increase of bcl-2 dependency upon ibrutinib treatment (Fig 1d). Addition of ibrutinib to venetoclax led to decreased phosphorylation of ITK in vivo (Fig 1e). Two patients suffering from r/r T-PLL after failing at least two treatment lines including alemtuzumab were treated with the combination of venetoclax and ibrutinib resulting in significant clinical responses with substantial drops in leukocytosis and LDH as well as substantial clinical improvement (Fig 2a, b). The dynamic BH3 profiling in samples taken from these patients confirmed that the addition of ibrutinib is indeed increasing overall priming and Bcl-2 dependency (Fig 2c, d). Conclusion Our findings suggest efficacy of combinatorial treatment of venetoclax with ibrutinib in T-PLL. Mechanistically, ibrutinib dephosphorylated ITK in T-PLL cells and, furthermore, enhanced BCL2 dependency, both, in-vivo and in-vitro. Patients treated with the combination venetoclax and ibrutinib experienced profound clinical responses which needs further evaluation in an prospective clinical study on a larger cohort of r/r T-PLL patients. Disclosures Herbaux: Abbvie: Honoraria; Janssen: Honoraria; Takeda: Honoraria; BMS: Honoraria; Gilead: Honoraria. Mayerhoefer:Siemens: Research Funding, Speakers Bureau; BMS: Speakers Bureau. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; Research to Practice: Honoraria. Staber:Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ibrutinib - BTK inhibitor Venetoclax - bcl2 inhibitor

HDAC Inhibitor AR-42 Decreases CD44 Expression and Sensitizes Myeloma Cells to Lenalidomide

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3377-3377 ◽  
Author(s):  
Hector Cordero-Nieves ◽  
Douglas W. Sborov ◽  
Alessandro Canella ◽  
Zhongfa Liu ◽  
Jessica Consiglio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Single Agent ◽  
Advisory Committees ◽  
Cd44 Expression

Abstract Introduction: The first FDA-approved deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA, Vorinostat), was shown to be effective in vitro by a number of anti-neoplastic mechanisms. Despite minimal single-agent activity in multiple myeloma (MM), phase 1b studies combining HDACi’s with bortezomib salvaged some relapsed patients and prolonged progression free survival (PFS) from days (Vorinostat) to months (Panobinostat), albeit at the cost of significant side effects including fatigue, nausea, and vomiting. Phase 1/2 studies in combination with lenalidomide have demonstrated tolerability and activity in lenalidomide-refractory patients, but randomized trials are lacking. In MM, the anti-neoplastic mechanism of action for HDACi’s is unknown, but at biologically achievable concentrations, it has been theorized that they sensitize MM cells to other drugs by interfering with cell adhesion mediated drug resistance (CAM-DR), primarily involving the integrins CD44, CD49d (VLA-4), CD54 (ICAM-1), and/or CD184 (CXCR4). AR-42 (ARNO Therapeutics) is a novel orally bioavailable phenylbutyrate-based class I/II HDAC inhibitor that has greater anti-proliferative effects compared to Vorinostat in vitro and in vivo. It has been previously shown that in MM cell lines, AR-42 down-regulates the expression of gp130, and inhibits IL-6 induced activation of STAT3 and downstream targets including BCL-XL and Cyclin-D1, with minimal effects on the PI3K/AKT and MAPK pathways. CD44 is a type I transmembrane glycoprotein, which is directly transcribed by β-catenin, and its role in cell adhesion-mediated drug resistance (CAM-DR) for MM as well as other cancers has been largely investigated. Recent published data have shown that CD44 forms a complex with STAT3 and p300 (acetyltransferase) causing STAT3 activation in a cytokine- and growth factor-independent manner, and that CD44 over-expression is one of the main molecular mechanisms that contributes to Lenalidomide resistance in MM cells. Hypothesis: We hypothesize that CD44 down-regulation, both surface expression on MM cells as well as the soluble form in the blood, is the primary effect of AR-42 at concentrations achievable in humans, explaining its weak single agent effect and its improvement when combined with other therapeutic agents in the clinic. Methods: As part of a single center, dose-escalating, first-in-man phase 1 trial of single agent oral AR-42 administered orally three times weekly in 28-day cycles (3 weeks of treatment followed by a 7-day off treatment period), patients were accrued at 20-70 mg TIW in a standard 3+3 cohort design. Using peripheral blood obtained during cycle 1 of therapy, nCounter® GX Human Immunology assays and nCounter miRNA expression profile was performed to assess differentially expressed genes after AR-42 treatment. Enzyme-linked immunoabsorbent assay (ELISA), qRT-PCR and luciferase assays were also performed.To examine whether AR-42 treatment could sensitize the cells to Lenalidomide in vivo, we used GFP+/Luc+ MM.1S cells engrafted in NOD-SCID mice. Results: AR-42 in relapsed MM showed no confirmed partial responses, but did result in marginal responses in 3 out of 13 MM patients. We found that AR-42 in MM cells modulates the expression of many genes coding for surface receptors including CD44, CD48, CD46 and TRAF5 and affects the expression of several miRNAs. Our data show a decrease of CD44 mRNA expression in the CD138+ MM plasma cells and of the soluble CD-44 in the serum of AR-42 treated patients. We also show that in MM cells CD44 down-regulation upon AR-42 treatment is associated with impairment of STAT-3 signaling pathways and direct targeting of its regulatory RNA binding protein, Insulin grow factor 3 binding protein 3 (IGF2BP3), by miR-9-5p. We found that miR-9-5p is up-regulated in vitro and in the cancer cells of MM patients after AR-42 treatment. Moreover we show that AR-42 in combination with Lenalidomide show synergistic apoptotic effect on MM cells, enhancing Lenalidomide anti-myeloma activity invivo. Conclusions: These findings show that CD44 is a therapeutic target for the HDACi AR-42 in MM patients, providing the rationale to support further clinical investigation of AR-42 in combination with IMiDs in patient cohorts with high pretreatment CD44 expression in the serum and on the surface of MM cells. Disclosures Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Donor-Derived Adoptive T-Cell Therapy Targeting Multiple Tumor Associated Antigens to Prevent Post-Transplant Relapse in Patients with ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 471-471
Author(s):  
Swati Naik ◽  
Spyridoula Vasileiou ◽  
Ifigeneia Tzannou ◽  
Manik Kuvalekar ◽  
Ayumi Watanabe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Management Support ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Post Infusion ◽  
T Cell Lines

Abstract Background: Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk Acute Lymphoblastic Leukemia (HR-ALL), but relapse remains a major cause of treatment failure. Strategies to enhance the graft-versus-leukemia (GVL) effect have been employed to prevent relapse, including modulating immune suppression post-HSCT to hasten immune reconstitution or with the use of donor lymphocyte infusions (DLIs). However, DLIs carry a significant risk of graft-versus-host disease (GVHD) due to the concurrent transfer of alloreactive T cells. To enhance the GVL effect while minimizing GVHD, we developed a protocol for the generation of ex vivo expanded, donor-derived T-cell lines targeting PRAME, WT1 and Survivin - tumor associated antigens that are frequently expressed in both B- and T-cell ALL. These multi-antigen-targeted T cells (multiTAAs) were adoptively transferred to pediatric and adult patients with HR-ALL who had undergone an allogeneic HSCT. Methods: Donor-derived multiTAA-specific T cells were generated by co-culturing PBMCs with autologous DCs loaded with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. Following 2-4 rounds of stimulation these multiTAA-specific T cells were infused to patients with ALL who had undergone an HSCT but remained at a high risk for disease relapse. Results: We have generated 15 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 95.1±1.9%) with a mixture of CD4+ (mean 22.8±6.3%) and CD8+ (mean 52.5±5.3%) cells, which expressed central [CD45RO+/CD62L+: 13.5±2.8%] and effector memory markers [CD45RO+/CD62L-: 56.4±3.8%]. The expanded lines recognized the targeted antigens PRAME (range 0-370 SFC/2x10 5), WT1 (0-363 SFC/2x10 5), and Survivin (0-65 SFC/2x10 5) in an IFNg ELIspot. None of the lines reacted against non-malignant patient-derived cells (3.7±0.8% specific lysis; E: T 20:1) - a study release criterion indicating lack of alloreactivity. We have infused 11 HR-ALL patients (8 pediatric and 3 adult) with donor-derived multiTAA-specific T cells to prevent disease relapse (Table 1). Patients were administered with up to 4 infusions of cells at 3 escalating dose levels, ranging from 0.5 - 2x10 7 cells/m 2. Infusions were well tolerated with no dose-limiting toxicity, GVHD, cytokine release syndrome or other adverse events. Three patients were not evaluable per study criteria as they received >0.5mg/kg of steroids (2 patients received stress doses for septic shock and 1 for elevated liver enzymes presumed to be GVHD that was later ruled out) within 4 weeks of infusion and were replaced. Six of the 8 remaining patients infused remain in CR on long-term follow up at a median of 46.5 months post-infusion (range 9-51 months). In patients who remained in long term CR we detected an expansion of tumor-reactive T cells in their peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading, which correlated temporally (within 4 weeks) with multiTAA infusions. By contrast in the two patients who relapsed we saw no evidence of in vivo T cell amplification within the first 4 weeks after infusion. Conclusion: The preparation and infusion of donor-derived multiTAA-specific T cells to patients with B- and T-ALL post allogeneic HSCT is feasible, safe and as evidenced by in vivo tumor-directed T cell expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Figure 1 Figure 1. Disclosures Vasileiou: Allovir: Consultancy. Tzannou: Gileas: Honoraria; Allovir: Current equity holder in publicly-traded company. Kuvalekar: Allovir: Consultancy. Watanabe: Allovir: Consultancy. Grilley: QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support; Allovir: Current equity holder in publicly-traded company, Other: Leadership. Hill: Incyte: Membership on an entity's Board of Directors or advisory committees. Omer: Allovir: Research Funding. Gottschalk: Tessa Therapeutics: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Tidal: Consultancy; Novartis: Consultancy; Catamaran Bio: Consultancy. Heslop: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Rooney: Allogene: Patents & Royalties; Bellicum: Patents & Royalties; Bluebird: Current equity holder in publicly-traded company; Allovir: Current equity holder in publicly-traded company; Alimera: Consultancy; Memgen: Consultancy; TScan Therapeutics: Consultancy; Takeda: Patents & Royalties; Marker: Current equity holder in publicly-traded company; Tessa: Consultancy, Other: Leadership, Research Funding. Vera: Allovir: Consultancy, Current equity holder in publicly-traded company, Other: Leadership, travel , accomodations, expenses, Patents & Royalties; Marker: Current Employment, Other: Travel, Accomodations, Expenses, Patents & Royalties, Research Funding. Leen: Allovir: Consultancy, Current equity holder in publicly-traded company; Marker: Current equity holder in publicly-traded company.

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