scholarly journals Venetoclax Synergizes with the RNA-Directed Nucleoside Analog 8-Chloro-Adenosine in Acute Myeloid Leukemia in Vitro and In Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Ralf Buettner ◽  
Le Xuan Truong Nguyen ◽  
Corey James Morales ◽  
Lisa S Chen ◽  
Timothy Synold ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Single Agent ◽  
Inhibitory Effect ◽  
Nucleoside Analog ◽  
Research Support ◽  
Advisory Committees

Relapse of acute myeloid leukemia (AML) is attributed to the persistence of quiescent leukemia stem cells (LSCs). Bcl-2 inhibition has been shown to target primitive leukemia progenitors. Venetoclax (VEN) is a FDA-approved Bcl-2-selective inhibitor for the treatment of AML. Although the activity of single agent VEN in AML patients (pts.) is modest, clinical efficacy in newly diagnosed, older pts. unfit for intense chemotherapy has been shown when VEN is combined with the hypomethylating agents (HMAs) azacytidine and decitabine or with low-dose nucleoside analog cytarabine. We have recently shown that VEN in combination with HMAs augments oxidative stress in AML cells and provided a molecular mechanism for the VEN-HMA-regulated NF-E2-related factor 2 (Nrf2) antioxidant pathway that could explain the results observed in early clinical studies in AML. Although about 70% of pts. initially respond to these VEN treatment regimens, about 30% of pts. do not and diminished efficacy of VEN combination treatments have been observed in pts. harboring poor-prognosis markers such as FLT3-ITD. In addition, future relapse of a percentage of pts. treated with VEN combinations is expected. Thus, novel treatment options for are urgently needed. We previously reported that the ribose containing, RNA-directed nucleoside analog 8-chloro-adenosine (8-Cl-Ado) demonstrates cytotoxic activity against AML cells and LSCs in vitro and in vivo, without significantly affecting normal hematopoietic stem cells. Importantly, our initial, unpublished results from a phase I/II clinical trial with single agent 8-Cl-Ado in pts. with refractory/relapsed AML demonstrate encouraging clinical benefits. Moreover, we have reported that FLT3-ITD AML is particularly sensitive to 8-Cl-Ado, thus suggesting 8-Cl-Ado plus VEN as a potential novel therapeutic regimen for treatment of AML. We here report that the VEN plus 8-Cl-Ado combination inhibited in vitro growth and induced apoptosis in AML primary cells, LSCs and cell lines significantly more compared to treatment with the individual agents. For in vitro cell growth studies, combination indices of <1 for all experimental and calculated drug concentrations demonstrated strong synergy between the two drugs in 2 human AML cell lines (MV4-11 and KG-1a) and in AML cells isolated from 2 pts. Moreover, immune compromised NSG mice engrafted with FLT3-ITD MV4-11 cells survived significantly longer when treated with VEN (20 mg·kg‒1·day‒1, daily oral) plus 8-Cl-Ado (50 mg·kg‒1·day‒1; osmotic pump), as compared to single agent or vehicle-treated mice (p<0.006, VEN+8-Cl-Ado vs. 8-Cl-Ado; p<0.001 VEN+8-Cl-Ado vs. VEN). LSCs depend on amino acid metabolism-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for energy production. VEN is known to target LSCs through inhibition of OXPHOS by targeting amino acid uptake/metabolism. We report here that 8-Cl-Ado inhibited the FAO pathway and down-regulated the oxygen consumption rate (OCR), a marker for OXPHOS, in LSCs. However, whereas 500 nM of 8-Cl-Ado was sufficient to induce MV4-11 growth inhibition, 1 microM of 8-Cl-Ado was needed for maximum inhibitory effect on FAO. We also report that 8-Cl-Ado increased expression of the anti-apoptotic protein p53. It was previously reported that p53 induces FAO in LSCs. Knockdown of p53 by siRNA augmented the inhibitory effect of 8-Cl-Ado on FAO and OCR. Importantly, addition of VEN could completely overcome the p53-induced activation of FAO and OCR. Mechanistically, we show that 8-Cl-Ado inhibited ribosomal RNA (rRNA) synthesis, a prerequisite for cellular proliferation, through down-regulation of the transcription initiation factor 1 (TIF-IA) protein. Since TIF-1A negatively regulates p53 expression, the inhibition of TIF-1A by 8-Cl-Ado resulted in up-regulation of p53 and subsequent p53-induced upregulation of FAO and OCR, thus diminishing the suppressive effects of 8-Cl-Ado on FAO and OCR. We further show that the VEN plus 8-Cl-Ado combination strongly induced p53 signaling, as shown by activation and inhibition of downstream p21 and PCNA proteins, respectively. This combination also augmented DNA fragmentation and apoptosis in LSCs. Thus, our data suggest that the synergy seen in AML with the VEN plus 8-Cl-Ado combination can be explained at least in part due to augmented inhibition of FAO and OXPHOS and represents a promising novel treatment for AML. Disclosures Pullarkat: Dova: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marcucci:Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial). Rosen:Celgene: Speakers Bureau; NeoGenomics: Consultancy; Seattle Genetics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; Pebromene: Consultancy.

Phase I Study of IMGN901, Used as Monotherapy, in Patients with Heavily Pre-Treated CD56-Positive Multiple Myeloma - A Preliminary Safety and Efficacy Analysis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2883-2883 ◽  
Author(s):  
Asher Chanan-Khan ◽  
Jeffrey Wolf ◽  
Mecide Gharibo ◽  
Sundar Jagannath ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
Board Of Directors ◽  
Dose Level ◽  
Single Agent ◽  
Duration Of Treatment ◽  
Advisory Committees ◽  
Grade 3

Abstract Abstract 2883 Poster Board II-859 Background: IMGN901 (huN901-DM1/BB-10901) is a novel anticancer agent consisting of a potent cytotoxic maytansinoid, DM1, attached to a CD56-binding monoclonal antibody, huN901, using an engineered linker. Once bound to CD56 on a cancer cell, the conjugate is internalized and releases DM1. About 70% of multiple myeloma (MM) cases have surface expression of CD56. In preclinical settings, IMGN901 showed significant in vitro and in vivo anti-myeloma activity as a single agent and in combination with approved drugs such as lenalidomide. Objectives: To determine the maximum tolerated dose (MTD), pharmacokinetics (PK), and activity of IMGN901, used as monotherapy, in patients with MM. Methods: Patients with CD56+ relapsed or relapsed/refractory MM receive a single IV infusion of IMGN901 on 2 consecutive weeks every 3 weeks. Patients are enrolled into each dose level in cohorts of 3, with dose-limiting toxicity (DLT) triggering cohort expansion. The European Bone Marrow Transplant (EBMT) criteria were used for response assessment. Results: Twenty-three CD56+ MM patients have received IMGN901 at doses ranging from 40 to 140 mg/m2/week. Most of these 23 patients had been treated with 6 or more chemotherapy regimens prior to study entry. Two of 6 patients treated at the 140 mg/m2/week dose experienced DLT (grade 3 fatigue and grade 3 acute renal failure) and a lower dose has been defined as the MTD. Commonly reported adverse events that were at least possibly related to IMGN901 were fatigue, increased aspartate aminotransferase, increased uric acid, sensory neuropathy and headache. None of the patients experienced serious hypersensitivity reactions or demonstrated a humoral response against either the antibody or DM1 component of IMGN901. Sustained partial response (PR) was documented in 1 patient treated at 140 mg/m2/week and 3 minor responses (MR) were reported in 1 patient each at doses of 60, 90, and 112 mg/m2/week. Of the 23 patients receiving any dose level of IMGN901, 8 remained on IMGN901 treatment for at least 15 weeks. Five of these 8 patients continued treatment on IMGN901 for at least 24 weeks, and two of these 5 patients remained on IMGN901 for at least 50 weeks. Preliminary PK results indicate an approximately linear relationship between dose and observed maximal serum concentration. Conclusion: This is the first study of IMGN901 in patients with MM. The MTD of this agent in MM patients is now defined. Our experience with IMGN901 in this clinical trial demonstrates an overall favorable safety profile. Although the primary objective of this clinical trial was to determine the MTD of single agent IMGN901, exciting single agent activity was observed in heavily pretreated MM patients. This is particularly encouraging as the duration of treatment with IMGN901 in some patients was longer than duration of treatment with prior regimens of approved agents. Clinical observations noted here (including single agent efficacy and the favorable toxicity profile) as well as findings from preclinical combination studies warrant continued investigation of this novel agent in patients with MM especially in combination with approved anti-myeloma agents/regimens such as lenalidomide and dexamethasone. Disclosures: Chanan-Khan: 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; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Merck: Honoraria. Miller:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Guild:ImmunoGen, Inc: Employment. Zildjian:ImmunoGen, Inc: Employment. Qin:ImmunoGen, Inc.: Employment. O'Leary:ImmunoGen, Inc.: Employment.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: 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. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4712-4712 ◽  
Author(s):  
Deepika Sharma Das ◽  
Ze Tian ◽  
Arghya Ray ◽  
Durgadevi Ravillah ◽  
Yan Song ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Clinical Trial ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

scholarly journals Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3298-3298
Author(s):  
Lili Feng ◽  
Haohai Zhang ◽  
Paola de Andrade Mello ◽  
Dina Stroopinsky ◽  
Wenda Gao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Lentiviral Vector ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Corresponding author: Dr. Simon. C. Robson ([email protected]). Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model. Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions. Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I). Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models. Disclosures: No relevant conflicts of interest to declare. References: [1] Nesrine Aroua, Emeline Boet, Margherita Ghisi, et al. Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia. Cancer Discov. 2020. Figure 1 Figure 1. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: 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; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

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

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

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

scholarly journals Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1451-1451
Author(s):  
Sigal Tavor ◽  
Tali Shalit ◽  
Noa Chapal Ilani ◽  
Yoni Moskovitz ◽  
Nir Livnat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Advisory Committees ◽  
Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p&lt;0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

Pooled Safety Analysis From Phase (Ph) 1 and 2 Studies of Carfilzomib (CFZ) In Patients with Relapsed and/or Refractory Multiple Myeloma (MM)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1954-1954 ◽  
Author(s):  
Seema B. Singhal ◽  
David Samuel diCapua Siegel ◽  
Thomas Martin ◽  
Ravi Vij ◽  
Michael Wang ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Single Agent ◽  
Study Drug ◽  
Underlying Disease ◽  
Tolerability Profile ◽  
Treatment Schedule ◽  
Advisory Committees

Abstract Abstract 1954 Background: Carfilzomib (CFZ) is a novel, highly selective epoxyketone proteasome inhibitor that produces potent and sustained proteasome inhibition both in vitro and in vivo. CFZ appears to lack many of the off-target activities frequently associated with bortezomib (BTZ). This lack of off-target activity may account for observed differences in tolerability seen with CFZ including lack of significant neuropathy and minimal neutropenia and diarrhea. To date, single agent CFZ has been evaluated in Ph 1 and 2 studies in >600 patients, and the vast majority of patients treated had relapsed and/or refractory (R/R) MM. In these settings, CFZ has demonstrated durable single-agent activity and was well-tolerated in patients with advanced stage disease with co-morbidities including baseline neuropathy or renal insufficiency. Here we present the results of parallel safety analyses of patients from four Ph 1 and 2 studies with CFZ. Materials and Methods: The present safety analyses were based on data accumulated from patients enrolled in the following trials: PX-171-003 A0 (R/R MM), PX-171-003 A1 (R/R MM), PX-171-004 (relapsed MM), and PX-171-005 (R/R MM with varying degrees of renal function). In all studies, the treatment schedule was based on a 28-day cycle, dosing CFZ QDx2 each week for 3 weeks (Days 1, 2, 8, 9, 15, 16) with 12 days of rest. Doses of CFZ ranged from 15–20 mg/m2 in cycle 1 (005 [15 mg/m2], 003 A0 and A1, 004 [20 mg/m2]). In three studies CFZ was escalated to 27 mg/m2 after the first cycle, as tolerated (003- A1, 004-BTZ naïve subset and 005). In PX-171-005, low-dose dexamethasone was added in the majority of patients. Results: CFZ was well-tolerated by patients across the 4 studies analyzed. The most common treatment-emergent adverse events (AEs) included fatigue, anemia, nausea, dyspnea, and thrombocytopenia. Detailed descriptions of the incidence of treatment-related AEs (all Grades (G) in ≥25% of pts; ≥G3 in ≥5% of pts) across studies are presented in the table. Peripheral neuropathy (PN) occurred infrequently across all 4 studies (N= 517), with only 20 patients (3.9%) experiencing PN of any G and only 2 patients (0.4%) with G3 PN. Febrile neutropenia was likewise uncommon, occurring in only 3 patients (0.6%). Serious treatment emergent AEs (SAEs) occurring in ≥1% of patients and considered possibly/probably related to study drug across all 4 studies included: pneumonia (3.5%), congestive cardiac failure (2.5%), acute renal failure (1.7%), pyrexia (1.2%), and dyspnea (1%). Conclusions: Despite a substantial disease burden present in the patient populations described here, CFZ was well-tolerated by patients with MM across all studies examined. The excellent safety/tolerability profile of CFZ has permitted prolonged administration (in some cases over 24 mos of continuous therapy including extension study) with minimal dose modifications or discontinuations due to toxicity. The low levels of neuropathy seen with CFZ make this agent a potentially important treatment option for patients with pre-existing neuropathy from either underlying disease or prior neuropathic anti-myeloma therapy. Disclosures: Singhal: Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding. Siegel:Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Martin:Celgene: Honoraria; Onyx: Consultancy. Vij:Onyx: Honoraria. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotec: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Kukreti:Celgene: Honoraria; Roche: Honoraria; Ortho Biotech: Honoraria. Zonder:Millenium: Consultancy, Honoraria, Research Funding; Cephalon: Research Funding; Celgene: Honoraria. Wong:Onyx Pharmaceuticals: Employment. McCulloch:Onyx Pharmaceuticals: Employment. Kauffman:Onyx Pharmaceuticals: Employment. Niesvizky:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millenium: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding. Stewart:Millennium: Consultancy; Celgene: Honoraria. Jagannath:Millenium, OrthoBiotec, Celgene, Merck, Onyx: Honoraria; Imedex, Medicom World Wide, Optum Health Education, PER Group: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Pharmacodynamic and Pharmacokinetic Properties of a Novel and Selective HDAC6 Inhibitor, ACY-1215, in Combination with Bortezomib in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2912-2912 ◽  
Author(s):  
Loredana Santo ◽  
Teru Hideshima ◽  
Andrew L. Kung ◽  
Jen-Chieh Tseng ◽  
David Tamang ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Tumor Tissue ◽  
Hdac Inhibitors ◽  
Histone H3 ◽  
Proteasome Inhibitors ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Aggresome Formation

Abstract Abstract 2912 HDAC enzymes are being studied as novel therapeutic targets in several cancers including multiple myeloma (MM). In particular, the combination of proteasome inhibitors (e.g. bortezomib (BZ)) with Histone Deacetylase (HDAC) inhibitors have shown very promising results in pre-clinical MM models. HDAC6, a Class II HDAC, has been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. Targeting both proteasomal and aggresomal protein degradation systems with proteasome inhibitors and HDAC inhibitors, respectively, induces accumulation of polyubiquitinated proteins, followed by activation of apoptotic cascades. Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with BZ. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC50 0.0054 mM) compared to the other HDACs, including Class I HDACs. Maximal cytotoxicity of ACY-1215 against MM cell lines was observed at 48h, with IC50 values ranging from 2–8 μM. To investigate the specific inhibitory effect of ACY-1215 on HDAC6 activity, we evaluated its effect on acetylation of a-tubulin. ACY-1215 induces potent acetylation of a-tubulin at low doses and triggers acetylation of lysine on histone H3 and histone H4 only at much higher doses, confirming its selective inhibitory effect on HDAC6 activity. Importantly, this selective inhibition was also observed in patient MM cells, where ACY-1215 increased acetylated a-tubulin after 4 h of treatment. We next combined low doses of ACY-1215 with BZ and showed synergistic anti MM activity, resulting in apoptosis via caspase-3, -8, -9 and poly (ADP) ribosome polymerase activation. Moreover, the combination of ACY-1215 plus BZ increased the accumulation of polyubiquitinated proteins compared to either agent alone. To investigate the effect of ACY-1215 on aggresome formation, MM.1S cells treated with ACY-1215 1 μM and/or BZ 2.5 nM for 12 h were stained with immunofluorescent anti-ubiquitin antibody. BZ-treated cells showed perinuclear structures consistent with aggresome formation, which was disrupted when BZ and ACY-1215 were combined. This result supports the synergistic anti MM activity of ACY-1215 with BZ. We also evaluated the in vivo anti-MM effect of combination therapy using two different xenograft models in SCID mice: plasmacytoma model and disseminated MM model. ACY-1215 in combination with BZ triggered more significant anti-MM activity than either agent alone in suppressing tumor growth and prolonging host survival in both models, without significant adverse effects. To optimize the design of future clinical trials, we conducted pharmacokinetic and pharmacodynamic studies in our plasmacytoma model. ACY-1215 peak plasma levels were observed at 4 h, which were unaffected by the addition of BZ. To further characterize the activity of ACY-1215 against HDAC6 in vivo, we evaluated the acetylation of α-tubulin in mouse blood cells by flow cytometry. The maximum levels of blood cell α-tubulin acetylation were observed at 4 h, providing an important biomarker for future clinical trials. Importantly, levels of acetylated α-tubulin were also detected in tumor tissue from treated mice in a similar time frame to peak blood levels, suggesting that ACY-1215 is readily absorbed by tumor tissue. Moreover, ACY-1215 did not accumulate in tumor tissue, as shown by the decline of acetylated α-tubulin in blood cells and tumor tissue by 24 h post-dose, which parallels the elimination of ACY-1215 from blood. We further confirmed the HDAC6 selectivity of ACY-1215 in our in vivo models by investigating the effect of the drug combination on histone acetylation in tumor tissue. WB analysis and IHC did not show a significant increase in acetylated histone H3 (lys 18), while demonstrating a robust acetylation of α-tubulin, the primary marker of HDAC6 inhibition by ACY-1215 at the cellular level. The results from our in vitro and in vivo studies therefore show significant and synergistic anti-MM activity of ACY-1215 in combination with BZ and provided the rationale for the ongoing phase I/II clinical trial in patients with relapsed or relapsed/refractory MM. Moreover, our pharmacodynamic helped inform the design of correlative studies, which will establish whether acetylated α-tubulin can be used as predictive biomarker of HDAC6 inhibition and disease response. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Ogier:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon: Consultancy. Anderson:celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Acetylon Pharmaceuticals Inc: founder. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

scholarly journals Hypomethylating Agent Therapy for Chronic Myelomonocytic Leukemia Does Not Impact Acute Myeloid Leukemia Transformation or Survival

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Sandrine Niyongere ◽  
Yamini Kathari ◽  
Zeba Singh ◽  
Emily J. Vannorsdall ◽  
Ashkan Emadi ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Chronic Myelomonocytic Leukemia ◽  
Institutional Research ◽  
Research Support ◽  
Advisory Committees ◽  
Clinical Trial Research ◽  
Myelomonocytic Leukemia

Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). CMML is characterized by persistent blood monocytosis &gt;1 x 109/L, bone marrow dysplasia in one or more hematopoietic cell lines, and increased risk of transformation to acute myeloid leukemia (AML). Our review of SEER Medicare data (Haematologica 2013;98:584) demonstrated that, compared to MDS, CMML has shorter overall survival (OS) and more frequent progression to AML. Hypomethylating agents (HMAs) have become standard therapy for CMML, with reported response rates of 37-69%, but their impact on AML transformation and OS is unclear. Methods: We retrospectively reviewed CMML patients treated at the University of Maryland Greenebaum Comprehensive Cancer Center between January 2000 and December 2019. Clinical characteristics, treatments, AML progression, time to AML progression (TTP), and OS were recorded and analyzed. Descriptive statistics were used for baseline characteristics and Kaplan-Meier analysis was performed for time-to-event data. Statistical analyses were performed using GraphPad Prism 8®. Results: We identified 71 patients with CMML, 82% male and 73% white, with a median age of 69 (range 25 - 96) years; 51% had &lt;10% bone marrow (BM) blasts and 45% had low-risk cytogenetic findings (normal karyotype or -Y). Most patients treated prior to 2005 received hydroxyurea and/or erythropoiesis-stimulating agents or were enrolled on clinical trials, while patients treated since 2005 received HMAs as primary therapy. Median follow-up was 41.1 months. The median OS of the entire cohort was 20 months, with 46% of patients progressing to AML with a median TTP of 11.5 months. By the MD Anderson Prognostic Scoring System at time of diagnosis, CMML was low-risk in 24 patients, intermediate-1 in 16, intermediate-2 in 14, and high-risk in 17. Forty-six patients received HMAs, with an overall response rate (ORR) of 54% (complete response or partial response), while 25 patients did not receive HMAs. Patient and disease characteristics were similar in HMA- and non-HMA-treated patients (Table 1). The estimated OS of HMA-treated patients was 20 months, compared to 14 months for non-HMA-treated patients (p =0.43) (Figure 1). AML transformation occurred in 52% of patients treated with HMAs, with TTP ranging from 3 to 65 months, and in 33% patients not treated with HMAs, with TTP ranging from 5 to 47 months. Most patients receiving HMAs (63%) received ≥ 6 cycles; 46% transformed to AML despite initial response, often in a sudden and unpredictable manner. HMAs were azacitidine in 13 patients, decitabine in 24, azacitidine followed by decitabine in 4, and decitabine followed by azacitidine in 5. Five CMML patients in our cohort underwent allogenic stem cell transplantation. Four of the five relapsed with transformation to AML post transplant, and only one patient remains in remission, 9 months post transplant. Conclusions: Despite a 54% ORR, HMA treatment did not have a significant impact on frequency of AML transformation, or OS in our cohort. Based on our data, favorable response rates previously reported with HMAs and also seen in our patients do not appear to translate into decreased frequency of AML transformation or prolonged OS. Though our study is a retrospective study with inherent selection bias, our results underscore the ongoing need for novel therapies and for clinical trials for CMML patients. Disclosures Niyongere: Kartos Therapeutics: Other: Received clinical trial research support with Kartos Therapeutics ; Forty Seven: Other: Received clinical trial research support with Forty Seven. Emadi:Amgen: Membership on an entity's Board of Directors or advisory committees; KinaRx: Other: co-founder and scientific advisor; NewLink Genetics: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Research Funding. Doung:Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: clinical trial research support; Incyte: Other: clinical trial research support; Astex: Other: clinical trial research support; MedPacto: Other: clinical trial research support. Baer:Takeda: Other: Institutional research funding; Oscotec: Other: Institutional research funding; Kite: Other: Institutional research funding; Incyte: Other: Institutional research funding; Forma: Other: Institutional research funding; Astellas: Other: Institutional research funding; AbbVie: Other: Institutional research funding.

scholarly journals Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2160-2160
Author(s):  
Gabriel Pabst ◽  
Johannes Foßelteder ◽  
Angelika Schlacher ◽  
Lisa Auinger ◽  
Daniel Martinez-Krams ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Genome Engineering ◽  
Specific Effect ◽  
Monocytic Differentiation ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays. Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition. Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed "Acute Myeloid Leukemia" among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells. Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. Figure 1 Figure 1. Disclosures Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

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