scholarly journals LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 50-51
Author(s):  
Raajit K. Rampal ◽  
John P. McGrath ◽  
Aishwarya Krishnan ◽  
Bing Li ◽  
Wenbin Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Myelogenous Leukemia ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees ◽  
Pathway Activation ◽  
Stat Pathway

Several novel mechanism-based therapeutic modalities are currently being clinically investigated for the treatment of patients with acute myelogenous leukemia (AML), including agents that exploit genomic vulnerabilities, attenuate leukemia stem cell populations and/or or synergize with anti-leukemic cytotoxic/epigenetic therapies. Lysine-specific demethylase 1 (LSD1) is an enzyme that functions in transcriptional repression by catalyzing the removal of histone H3 lysine 4 methylation, a histone modification associated with transcriptionally competent gene enhancers and transcriptional start sites. Small molecule mediated inhibition of LSD1 alters the chromatin state and the transcriptional output of LSD1 target genes. Transcriptional 'reprogramming' by LSD1 inhibitors either causes a direct impact on cell fate and/or renders malignant cells more susceptible to the treatment with other cancer therapeutic agents. LSD1 inhibitors have shown encouraging phenotypic effects in myelogenous leukemia (AML) models but the key molecular determinants governing LSD1 inhibitor sensitivity remain to be further investigated. Here, we explored the in vitro sensitivity of 350 cancer cell lines to our LSD1 inhibitor CPI-482 to identify potential hyper-responder cell contexts. Four AML cell lines showed high sensitivity with low nanomolar concentration GI50s, each of which contained either a JAK2V617F mutation or a genetic aberration that resulted in JAK-STAT pathway activation. Oral administration of LSD1 inhibitor CPI-482 on a once daily or a once weekly dosing schedule resulted in significant tumor growth inhibition in SET-2 and HEL 92.1.7 JAK2 mutant AML xenograft mouse models. Given the unmet need and poor prognosis in post-MPN secondary AML (sAML) we then explored CPI-482 in a tertiary transplant post-MPN AML retroviral transduction murine model (Jak2V617F retrovirus transduced intoTp53 null bone marrow). Jak2V617F/Tp53 null spleen cells were transplanted into lethally irradiated recipient mice along with wild-type donor support whole bone marrow cells. Mice were randomized to treatment with vehicle, Ruxolitinib (60mg/kg twice daily) or CPI-482 (60mg/kg weekly). Once-weekly treatment with CPI-482 significantly improved survival compared to vehicle (p<0.001) or ruxolitinib (p<0.043) (Figure 1A). Spleen weights were significantly reduced by CPI-482 compared to ruxolitinib (p<0.05;Figure 2B). The white blood cell count was unchanged in mice treated with CPI-482 but increased in both vehicle and ruxolitinib treated mice. Evaluation at the time of terminal take-down of mice demonstrated a significant increase in the proportion of lineage positive cells in both the bone marrow and spleen (compared to vehicle, p<0.05) consistent with restoration of normal hematopoiesis (Figure 1D). Histopathologic evaluation of the spleen demonstrated marked reduction in infiltration by blast cells, restoration of lymphoid follicles, emergence of megakaryocytes (Figure 1E), and modest reductions in reticulin fibrosis in CPI-482 treated mice, which was not observed in ruxolitinib treated mice. Mice tolerated treatment with CPI-482 well, with no changes in weights of treated mice (Figure 1F). Treatment of the JAK2V617F mutant AML cell lines SET-2 and HEL with CPI-482 resulted in specific transcriptional effects, including increased expression of the myeloid differentiation markers LY96 and CD86 and inflammatory response genes. CPI-482 also resulted in upregulation of genes that are repressed by the HOXA9 oncogene in other leukemia contexts. The induction of specific CPI-482 mediated gene expression and phenotypic changes was recapitulated by knockdown of the transcription factor GFI1B, suggesting that, consistent with prior findings in other leukemia contexts, LSD1 functionally cooperates with GFI1B in JAK2V617F mutant AML cells. These data provide support for a potential therapeutic impact of the LSD1 inhibitor CPI-482 in AML and sAML in the context of the JAK2V617F mutation, and thus extend the previous findings that LSD1 inhibitors may have utility in JAK2V617F mutated malignant proliferative neoplasms. Given the pressing need for new therapies for sAML which evolves from a pre-existing MPN, we believe these data form the rationale for a mechanism based clinical trial in this adverse risk myeloid malignancy. Figure Disclosures Rampal: Pharmaessentia: Consultancy; Galecto: Consultancy; Abbvie: Consultancy; Stemline: Consultancy, Research Funding; Constellation: Research Funding; Incyte: Consultancy, Research Funding; Celgene: Consultancy; Promedior: Consultancy; CTI Biopharma: Consultancy; Jazz Pharmaceuticals: Consultancy; Blueprint: Consultancy. McGrath:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Xiao:Stemline Therapeutics: Research Funding. Nikom:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wang:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Levine:Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Trojer:Constellation Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Dual CXCR4 and E-Selectin Inhibition (GMI-1359) Rapidly Increases AML Cellular Motility Prior to Intravasation and Vascular Niche Depletion Observed By Intravital Bone Marrow Two-Photon Microscopy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Tomasz Zal ◽  
Malgorzata Anna Zal ◽  
Mateusz Rytelewski ◽  
Kyung Hee Chang ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Cellular Motility ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Vascular Niche

BACKGROUND: Acute myelogenous leukemia (AML) cells that reside in bone marrow (BM) receive a great deal of protection from the cytotoxic effects of therapeutic agents. In contrast, the circulating leukemia cells are typically more chemosensitive compared to those embedded in BM niches. The BM homing of AML cells is mediated by multiple adhesive and chemokinetic interactions including, respectively, by sialylated glycoproteins on the cancer cells binding to E-selectin on the endothelium and by CXCR4-mediated sensing of SDF-1/CXCL12 gradients. Consequently, both E-selectin and CXCR4 inhibitors have been pursued as targets for add-on therapeutic strategies aimed to "mobilize" AML cells out of the BM. A next generation "mobilizing" agent GMI-1359 combines, in one molecular structure, the E-selectin and CXCR4 inhibitory moieties. In a previous study, GMI-1359 markedly reduced leukemia cell adhesion to endothelial cells, leukemia cellularity in BM (Zhang et al., 2016) and it enhanced the survival of cytarabine/daunorubicin therapy in a FLT3-mutated AML model (Zhang et al., 2015). APPROACH: Here, we used intravital 2-photon microscopy of calvarial BM to study the behavioral response of AML cells to the dual CXCR4 and E-selectin inhibition (GMI-1359) in vivo. We used a mTurquoise2 fluorescence-tagged transplantable mouse AML model (characterized by MLL, ENL-FLT3, ITD, p53-/-). To delineate the bone and vascular niches, the syngeneic immune-competent recipient mice harbored cell lineage fluorescence reporter genes such as Col2.3-GFP, hCD2-DsRed and CD11c-EYFP with the bone collagen and blood highlighted by, respectively, second harmonic generation (SHG) and fluorescent dextran. Intravenously infused AML cells (5x10E4) homed to BM where they gradually displaced most endogenous cells. In this experimental system, GMI-1359 or vehicle were infused into the tail vein while recording AML cell motilities in calvarial BM stroma in 3-D over 4 hours. RESULTS: In untreated or control vehicle treated mice, AML cells were slowly motile, migrating with the average velocity of ~2 µm/min. Cellular trajectories were random within the average 400 µm confinement radius corresponding to the size of BM cavities. Most AML cells localized in the vascular niche, defined as within a 50 µm distance to the nearest blood vessel. A minority of AML cells were also present in the proximity to osteoblasts and the bone (i.e. in the bone niche). In contrast, GMI-1359 infusion resulted in a 66% increase of the average speed of cellular motility of AML cells within 20 min and up to 100% increase within 3.5 hours. The motility pattern remained largely random within the bone cavity confinement volume and was concentrated in the vicinity of vasculature. Thereafter, we observed multiple instances of AML cell intravasation into the capillary lumens followed by several minute-long vessel wall attachment and sudden outflow. This cellular dynamics resulted in a substantial, yet structurally biased decrease of BM AML cellularity whereby the vascular niche was emptied preferentially whilst the bone niche remained populated by AML cells. In no case we observed a complete BM depletion of AML cells. Interestingly, as we reported before, a single, CXCR4-only inhibitor caused a protracted (24-48 h) depletion of AML cells in BM without significant cellular motility enhancement. CONCLUSIONS: Our observations reveal an unexpected mechanism of action by a dual E-selectin/CXCR4 inhibitor involving cellular migratory motility enhancement in BM stroma prior to AML cell intravasation. We envisage that, besides the designed blocking of E-selectin and CXCR4 molecules from binding their corresponding ligands, the dual moiety GMI-1359 agent has a capacity to generate motility-enhancing signals in AML cells that single inhibitors do not seem to trigger. Among several mechanisms possible, this action could involve E-selectin and CXCR4 crosslinking by a dual moiety molecular structure or binding avidity enhancement, requiring further investigation. In addition, our results highlight a likely mechanism of AML resistance to therapeutic "mobilization", one that involves cancer cell persistence in the bone/osteoblastic niches. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. 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; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: 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.

scholarly journals Genotype-Phenotype Relationships and Therapeutic Targets in Acute Erythroid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
June Takeda ◽  
Kenichi Yoshida ◽  
Akinori Yoda ◽  
Lee-Yung Shih ◽  
Yasuhito Nannya ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Private Company ◽  
Advisory Committees ◽  
Otsuka Pharmaceutical ◽  
Group A ◽  
Genetic Profiles ◽  
Stat Pathway

Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although gene mutations in AEL have been described in several reports, genotype phenotype correlations are not fully understood with little knowledge about the feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 105 AEL cases with the median age of 60 (23-86), using targeted-capture sequencing of commonly mutated genes in myeloid neoplasms, together with 1,279 SNPs for copy number measurements. Among these 105 cases, 13 were also analyzed by RNA sequencing. Genetic profiles of these 105 AEL cases were compared to those of 775 cases with non-erythroid AML (NEL) including 561 cases from The Cancer Genome Atlas and Beat AML study. An immature erythroid cell line (TF1) and three patient-derived xenografts (PDX) established from AEL with JAK2 and/or EPOR amplification. Cell line and samples from patients were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 subgroups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype and showed very poor prognosis. Remarkably, all PEL cases were categorized into Group A. Conspicuously, 80% of PEL cases had amplifications of JAK2 (6/10; 60%), EPOR (7/10;70%), and ERG (6/10;60%) loci on chromosomes 9p, 19q, and 21q, respectively, frequently in combination, although they were rarely seen in NEL cases. All cases in Group B (n=19, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. To further characterize this subgroup, we compared genetic profiles of STAG2-mutated AEL and NEL. Prominently, 70% (14/20) of STAG2-mutated cases in AEL had KMT2A-PTD, whereas it was found only in 8.8% (3/34) of NEL. CEBPA mutations were also more common in AEL (6/21; 29%) than NEL (4/34; 12%). While Group C was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in NEL, NPM1-mutated patents in this subgroup lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in NEL (25/209; 12%). The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, U2AF1 and KMT2C. Recurrent loss-of-function mutations in USP9X were unique to this subtype, although USP9X mutations have been reported in ALL with upregulation of JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only those cases with JAK2 or EPOR amplification, but also those without, suggesting that aberrantly upregulated STAT5 activation might represent a common defect in AEL. Based on this finding, we evaluated the effect of a JAK inhibitior, ruxolitinib, on an AEL-derived cell line and three PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest, ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with TF1 and 2 PDX models with STAT5 downregulation, although the other model was resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2, EPOR and/or ERG. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a role of JAK inhibition was suggested. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Miyazaki:Astellas Pharma Inc.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Takaori-Kondo:Celgene: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; MSD: Honoraria. Kataoka:Asahi Genomics: Current equity holder in private company; Otsuka Pharmaceutical: Research Funding; Takeda Pharmaceutical Company: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding. Usuki:Alexion: Research Funding, Speakers Bureau; Apellis: Research Funding; Novartis: Research Funding, Speakers Bureau; Chugai: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Ganser:Novartis: Consultancy; Celgene: Consultancy. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Ruxolitinib is used for drug efficacy test using patient-derived xenografts established from acute erythroid leukemia.

scholarly journals Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3686-3686
Author(s):  
Paul Brent Ferrell ◽  
William Senapedis ◽  
Alexander Cook ◽  
Erkan Baloglu ◽  
Yosef Landesman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Allosteric Modulators ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals FLT3 and LSD1 Inhibitor Combinations Synergistically Repress Growth of FLT3-Mutant Acute Myeloid Leukemia Via Blockage of MYC Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Daniel J. Coleman ◽  
Brittany M. Smith ◽  
Cody Coblentz ◽  
Rowan L. Callahan ◽  
Jake VanCampen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Transcriptional Activation ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Target Genes ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees ◽  
Flt3 Inhibitor

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Mao Yu Peng ◽  
Yasmin Abaza ◽  
Martina Mcdermott ◽  
Monica Mead ◽  
Dennis J. Slamon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Synergistic Effect ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
Single Agent ◽  
Therapeutic Strategies ◽  
Private Company ◽  
Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates >85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

scholarly journals Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Christian Marinaccio ◽  
Praveen K Suraneni ◽  
Hamza Celik ◽  
Andrew Volk ◽  
Jeremy Q. Wen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Private Company ◽  
Blast Phase ◽  
Advisory Committees ◽  
Self Renewal ◽  
Stat Signaling

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

scholarly journals Improved Gene Therapy for Metachromatic Leukodystrophy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3979-3979
Author(s):  
Lucas Tricoli ◽  
Adeline Vanderver ◽  
Laura Adang ◽  
Maxwell Chappell ◽  
Laura Breda ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Metachromatic Leukodystrophy ◽  
Mrna Translation ◽  
Advisory Committees ◽  
Patient Fibroblast

Abstract Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed. The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021. We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. Disclosures Vanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Genomic and Transcriptomic Characterization of IgM Multiple Myeloma Identifies a Pre-Germinal Center Plasma Cell Disorder with Immature B-Cell Transcription-Factor Signature

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Abdul Hamid Bazarbachi ◽  
Herve Avet-Loiseau ◽  
Zachary R Hunter ◽  
Raphael Szalat ◽  
Anil Aktas-Samur ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Switch Region ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees ◽  
Transcript Levels ◽  
Unique Signature

Multiple myeloma (MM) is a proliferation of terminally differentiated plasma cells (PC) producing monoclonal immunoglobulins (Ig), most commonly IgG and IgA (50% and 25% respectively), and less frequently, light-chain only disease, non-secretory, and IgD. IgM-MM is a rare entity (<0.5%), and its differentiation from common IgM producing PC disorders like Waldenström's macroglobulinemia (WM) is essential considering their distinct treatments and prognoses. Recent advancements in molecular techniques have shed light on the genomic characteristics and unique alterations in MM and WM, however, comprehensive profiling is still lacking for IgM-MM. We performed deep whole-genome sequencing on five IgM samples as well as 211 MM and 34 WM samples, and transcriptome sequencing on the IgM samples as well as 30 MM, 35 WM, and 3 PC. All IgM-MM samples harbored t(11;14) which combines super enhancers in Ig genes with CCND1. All translocations involved VHDHJH regions (Figure 1A) at the immunoglobulin heavy chain (IGH) locus, compared to IgG/IgA MM samples that had predominantly switch-region translocations (Figure 1B/C). Switch-region translocations are generated through class-switch recombination (CSR) in mature B-cells in germinal centers (GC), and VHDHJH translocations occur during recombination at the early pro-B-cell stage in the bone marrow (BM). While IgG/IgA-MM displayed evidence of CSR with deletions between IGHM switch-region and IGHG/IGHA switch regions, IgM-MM had no such events. IgM-MM therefore appears to undergo malignant transformation prior to late-stage B-cell maturation, after which CSR is unlikely, which is supported by a lack of progression of IgM-monoclonal gammopathy of undetermined significance (MGUS) to non-IgM-MM. IgM-MM also displayed similar copy number variation (CNV) patterns and driver mutations compared to non-IgM-MM suggesting similar progression events. Unsupervised hierarchical clustering using differentially expressed genes between non-IgM-MM and WM separated the IgM-MM samples within non-IgM-MM. This indicates a closer molecular homology to MM compared to WM with a unique signature for this group not accounted for by the t(11;14) translocation. Running the same analysis using only B-cell specific transcription factors (TFs) yielded similar results, with separation of WM and MM and preferential clustering of IgM-MM within the latter while also exhibiting a unique signature (Figure 1D). Some noteworthy examples were the upregulation of PBX3, PAX5, BCL11A, and ATF2, and the downregulation of PRDM1 and BCL3 compared to non-IgM-MM. The loss of PAX5 and upregulation of PRDM1 in B-cells has been implicated in promoting commitment to PC differentiation, while BCL11A was found essential for early B-cell progenitor development through the GC but extinguished in terminally differentiated PC. It appears that IgM-MM has therefore a more immature phenotype compared with non-IgM-MM, which further supports the previously discussed findings of its pre-GC origin and lack of terminal development. Three clinically relevant targets were noted to be upregulated in IgM-MM, Bruton's tyrosine kinase (BTK), CD20 and BCL-2. BTK was significantly higher in IgM-MM compared to non-IgM-MM (log2fold=1.3; FDR<10-3) with no difference between IgM-MM and WM (log2fold =-0.4; FDR>0.2). This could elucidate a more prominent role for BTK-inhibition in the IgM-MM subgroup. Furthermore, as documented in t(11;14)-MM, IgM-MM had elevated transcript levels of CD20 with possible targeting using anti-CD20 antibodies. Finally, elevated levels of BCL-2 in both IgM and non-IgM-t(11;14)-MM were observed, an established target for both single-agent and combination therapy. Interestingly, although not significant, IgM-MM had lower transcript levels of BCL-XL and MCL-1 compared to non-IgM-t(11;14)-MM, believed to be a predictor of higher sensitivity to venetoclax, and therefore an important guide for treatment choice. Clinical data however is lacking, and further investigations are needed to fully understand the potential role of these drugs in treating IgM-MM. In summary we describe a unique genomic and transcriptomic profile of IgM-MM, compared to both non-IgM-MM and WM, that describes its cellular origin and provides the rationale for potential therapeutic intervention. Disclosures Fulciniti: NIH: Research Funding. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: 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; 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; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Parmigiani:Phaeno Biotehnologies: Current equity holder in publicly-traded company; CRA Health: Current equity holder in publicly-traded company; Foundation Medicine Institute: Consultancy; Delphi Diagnostics: Consultancy; BayesMendel Laboratory: Other: Co-lead. Treon:Bristol-Meyer-Squibb: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding. Mohty:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Munshi:Takeda: Consultancy; BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy.

Patterns of Venetoclax Sensitivity in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-14
Author(s):  
James T Dibb ◽  
Nicola Long ◽  
Christopher A. Eide ◽  
Stephen E Kurtz ◽  
Cristina E. Tognon ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Single Agent ◽  
Drug Combinations ◽  
Lymphocytic Leukemia ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees

Patterns of Venetoclax Sensitivity in Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is predominantly a disease of older adults. The 5-year overall survival is 70-91%, depending on Rai/Binet stage at diagnosis (80% overall), and although a subset of CLL takes a very indolent course, it can be aggressive as well. Disease course and responsiveness to therapeutic agents may be predictable, to some degree, based on specific genetic lesions or other patient population characteristics. Monotherapies targeting specific cell pathways are rapidly increasing in prevalence. Ibrutinib (Bruton tyrosine kinase inhibitor) has shown promise as a single agent as well as in combination with other agents. In particular, ibrutinib has shown efficacy in combination with venetoclax (inhibitor of cell death suppressor BCL2). This combination appears to be particularly potent in patients with a del(11q) karyotype. Cytogenetic information is used already in several other leukemias to inform prognosis and treatment. Although CLL is a disease of monoclonal proliferation, precise definition of the diseased clone will allow for more individualized treatment. Stratification of drug sensitivity based on genetic and cytogenetic features will directly affect patient outcomes in CLL. Primary patient mononuclear cells (from either peripheral blood or bone marrow) were plated ex vivo with a panel of 49 drug combinations and the 16 respective single agents (SA) in 384-well plates using 10,000 cells/well. Drugs were tested in 7-point concentration series; wells with drug combinations were added at fixed molar ratios. Cell viability was assessed after a 72 hour culture period. In this assay, primary cells maintain viability but do not proliferate. In CLL, the most frequent mutations were: del(17p); del(11q); del(13q14); trisomy 12; complex karyotype (at least three chromosomal aberrations). Selected analysis of these data from 157 unique patients were performed by isolating the most potent inhibitors (defined by lowest median AUC) either as a single agent or in combination with known treatments. These were evaluated with nonparametric tests (Kruskal-Wallace, Mann-Whitney, Spearman rank coefficient) on the statistical software Prism. By subdividing the data by available genetic and cytogenetic information, patterns that have not been previously described in the literature emerged. In the cohort of patients with any karyotypic abnormality (not complex karyotype), SA venetoclax and the combination of venetoclax-ibrutinib (VEN/IBRUT) were equivalently effective with no significant difference in efficacy observed between SA venetoclax and the combination. As previously described, del(11q) independently predicts increased efficacy of SA venetoclax and VEN/IBRUT, and this efficacy was validated by ex vivo potency here as well. However, we show that male gender is an independent predictor of potency in both SA venetoclax and VEN/IBRUT as well. Interestingly, doramapimod (an inhibitor of p38 MAP kinase) was not particularly potent as a SA, however, the combination of venetoclax-doramapimod (VEN/DORA) proved to be the most potent of all combinations tested, more potent than even VEN/IBRUT. This effect could not be replicated in any subgroup, as VEN/DORA samples for the entire cohort were relatively limited (n=31). Although this analysis has inherent limitations, including underpowered data to analyze in less frequent cytogenetic events (e.g. del(6q)), we did find significant patterns of potency. These may or may not translate to clinical efficacy in CLL and do not address any potential toxicity. However, these data suggest future directions for more targeted research on these drugs and drug combinations. Disclosures Tyner: Petra:Research Funding;Janssen:Research Funding;Seattle Genetics:Research Funding;Incyte:Research Funding;Genentech:Research Funding;Constellation:Research Funding;AstraZeneca:Research Funding;Aptose:Research Funding;Gilead:Research Funding;Takeda:Research Funding;Syros:Research Funding;Agios:Research Funding;Array:Research Funding.Druker:EnLiven:Consultancy, Research Funding;Gilead Sciences:Consultancy, Membership on an entity's Board of Directors or advisory committees;Cepheid:Consultancy, Membership on an entity's Board of Directors or advisory committees;Dana-Farber Cancer Institute:Patents & Royalties;Bristol-Myers Squibb:Research Funding;Blueprint Medicines:Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Aptose Therapeutics Inc. (formerly Lorus):Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;ARIAD:Research Funding;Third Coast Therapeutics:Membership on an entity's Board of Directors or advisory committees;The RUNX1 Research Program:Membership on an entity's Board of Directors or advisory committees;Pfizer:Research Funding;Patient True Talks:Consultancy;Oregon Health & Science University:Patents & Royalties;Novartis Pharmaceuticals:Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding;MolecularMD (acquired by ICON):Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;Millipore (formerly Upstate Biotechnology):Patents & Royalties;VB Therapeutics:Membership on an entity's Board of Directors or advisory committees;Vivid Biosciences:Membership on an entity's Board of Directors or advisory committees;ALLCRON:Consultancy, Membership on an entity's Board of Directors or advisory committees;Amgen:Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees;Aileron Therapeutics:Membership on an entity's Board of Directors or advisory committees;Merck & Co:Patents & Royalties;McGraw Hill:Patents & Royalties;GRAIL:Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Henry Stewart Talks:Patents & Royalties;Iterion Therapeutics (formerly Beta Cat Pharmaceuticals):Membership on an entity's Board of Directors or advisory committees;Leukemia & Lymphoma Society:Research Funding.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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