scholarly journals A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3512-3512
Author(s):  
Manit Munshi ◽  
Xia Liu ◽  
Amanda Kofides ◽  
Nickolas Tsakmaklis ◽  
Maria G Demos ◽  
...  
Keyword(s):  
B Cell ◽  
Family Member ◽  
Board Of Directors ◽  
Research Funding ◽  
Marked Reduction ◽  
Wild Type ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Survival Signaling

Abstract Activating mutations in MYD88 (MYD88 Mut) are common in B-cell malignancies including Waldenstrom Macroglobulinemia (WM) and ABC subtype of diffuse B-cell lymphoma (ABC DLBCL). MYD88 is a component of the Toll-like receptor (TLR) pathway. We and others previously showed that MYD88 Mut triggers assembly of a "Myddosome" complex that leads to downstream pro-survival signaling that includes IRAK4/IRAK1 and BTK triggered NF-κB (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013) and HCK mediated BTK/NF-κB, PI3K/AKT, and MAPK/ERK signaling (Yang et al, Blood 2016; Liu et al Blood Adv. 2020). The activation of the B-cell receptor (BCR) signaling component SYK has also been observed in MYD88 Mut WM (Argyropoulos et al, Leukemia 2016). In ABC DLBCL, chronic active BCR signaling underlies SYK activation that is triggered by the SRC family member LYN (Davis et al, Nature 2010). These observations led us to explore potential drivers of BCR/SYK activation in WM. We previously reported that MYD88 Mut triggered activation of SYK in WM and ABC DLBCL cells (Munshi et al, BCJ 2020). Herein, we investigated if HCK, a SRC family member that is transcriptionally upregulated and activated by MYD88 Mut could trigger the BCR pathway through SYK activation. Since LYN is an integral part of BCR signaling, we first examined its expression and activation state in MYD88 Mut WM and ABC DLBCL cells. While MYD88 Mut TMD8, HBL-1 and OCI-Ly3 ABC DLBCL cells showed strong expression of p-LYN, such expression was absent or low in MYD88 Mut BCWM.1 and MWCL-1 cells, as well as CD19-selected bone marrow derived primary lymphoplasmacytic cells (LPCs) from WM patients. In view of the above findings, we next interrogated a direct role for HCK in mediating SYK activation. We over-expressed wild-type HCK (HCK WT) or gatekeeper mutated HCK (HCK T333M) in MYD88 Mut BCWM.1 and MWCL-1 WM cell lines, and TMD8 ABC DLBCL cells. In all these cell lines, over-expression of HCK WT or HCK T333M triggered a robust increase in phosphorylation of SYK Y525/Y526 in comparison to vector only transduced cells. Moreover, using an inducible vector system, knockdown of HCK showed a marked reduction in phosphorylation of SYK Y525/Y526 in MYD88 Mut BCWM.1 WM and TMD8 ABC DLBCL cells. We next sought to clarify if HCK and activated SYK were present in the same signaling complex. We performed co-immunoprecipitation experiments using an HCK antibody in MYD88 Mut BCWM.1, TMD8 and wild-type MYD88 (MYD88 WT) Ramos cells. The HCK antibody effectively pulled down p-SYK in MYD88 Mut BCWM.1 and TMD8 cells, but not in MYD88 WT Ramos cells. To confirm whether SYK activation was a result of HCK kinase activity, we next performed rescue experiments with the HCK inhibitors A419259 and KIN-8194 in MYD88 Mut BCWM.1 and MWCL-1 WM and TMD8 ABC DLBCL cells expressing either HCK WT or the HCK T333M protein that abrogated the activity of these inhibitors against HCK. Expression of the HCK T333M protein produced marked resistance to A419259 as well as KIN-8194 versus vector or HCK WT transduced BCWM.1 and MWCL-1 cells. By PhosFlow analysis, we observed that expression of HCK T333M but not HCK WT led to persistent activation of HCK and SYK in the presence of A419259 or KIN-8194 in BCWM.1 and MWCL-1 WM cells, and TMD8 ABC DLBCL cells. Consistent with these observations, treatment of primary MYD88 mutated WM LPCs cells with either A419259 or KIN-8194 also showed marked reduction in both p-HCK and p-SYK expression by PhosFlow analysis. Taken together, our findings show that SYK is activated by HCK in MYD88 Mut B-cell lymphomas cells; broaden the pro-survival signaling generated by aberrant HCK expression in response to MYD88 Mut; and help further establish HCK as an important therapeutic target in MYD88 Mut B-cell lymphomas. Disclosures Palomba: Juno: Patents & Royalties; Rheos: Honoraria; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Notch: Honoraria, Other: Stock; Kite: Consultancy; Novartis: Consultancy; BeiGene: Consultancy; Priothera: Honoraria; Nektar: Honoraria; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; WindMIL: Honoraria; Magenta: Honoraria; Pluto: Honoraria; Lygenesis: Honoraria; Ceramedix: Honoraria. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Gray: Syros, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi: Research Funding. Munshi: Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy. Anderson: Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: 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; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Yang: Blueprint Medicines Corporations: Current Employment, Current holder of individual stocks in a privately-held company. Treon: BeiGene: Consultancy, Research Funding; Eli Lily: Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding.

IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Shanye Yin ◽  
Gregory Lazarian ◽  
Elisa Ten Hacken ◽  
Tomasz Sewastianik ◽  
Satyen Gohil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Binding ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Experimental Group

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

scholarly journals Chronic Lymphocytic Leukemia B Cells Display IgM and IgD Isotype-Restricted Features That Affect Association with Co-Receptors, BCR Signaling, and Leukemic B-Cell Growth In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3124-3124
Author(s):  
Andrea Nicola Mazzarello ◽  
Marcus Dühren-von Minden ◽  
Eva Gentner ◽  
Palash Chandra Maity ◽  
Gerardo Ferrer ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Cell Size ◽  
Metabolic Activity ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Bcr Signaling

Abstract The leukemic cells in patients with chronic lymphocytic leukemia (CLL) are highly dependent on B-cell receptor (BCR) mediated signaling. Despite this and the fact that >90% of CLL clones co-express IgM and IgD, the composition and molecular mechanisms regulating BCR signaling regarding the two isotypes and the co-receptors with which they associate is lacking. Here we have addressed these issues. First, using Imaging Flow Cytometry, we evaluated BCR organization on the surface membrane of CLL cells from 11 patients who had participated in a 2H2O-labeling study that determined in vivoCLL B-cell birth rates (BR). We found that in all cases mIgM resided in more and larger surface clusters than mIgD. Also, a statistically significant, direct correlation was observed for IgM density and in vivoCLL-cell BR, with patients exhibiting more recently-divided cells having the highest expression of IgM. This was not the case for IgD. BCR signaling requires co-receptors that can co-localize differently with the two isotypes. Thus, we tested co-localization of stimulatory (CD20) and inhibitory (CD22) co-receptors with mIgM and mIgD, using the proximity ligation assay technique that discriminates 10 to 40 nm distances. Higher IgM:CD20 and lower IgD:CD20 co-localization ratios directly associated with in vivo BR. Conversely, patients whose CLL B cells showed greater IgM to CD22 co-localization ratios had lower BRs. Thus, association of IgM with stimulatory versus inhibitory co-receptors correlated with positive or negative regulation of CLL growth in vivo. Next, we questioned the extent that the observed differences in BCR organization affected the entire clone by measuring a marker of single cell metabolic activity - cell size. IgM and BR associated with entire clonal populations that were skewed toward larger, more active cells. Similarly, high BR CLLs displayed an increased mitochondrial maximal respiration and glycolytic activity and capacity, based on measurements of oxygen consumption rate and extracellular acidification rate, respectively. Since our findings supported a link between IgM- but not IgD-BCRs, growth rate in vivoand clonal metabolic activity, we questioned whether intrinsic, constitutive CLL BCR autonomous signaling differed for these two isotypes. To address this, we examined the signaling capacities of CLL-derived BCRs expressed as IgM or IgD isotypes, while maintaining the original IGHV-D-J and IGLV-J rearrangements. We used B cells that do not express endogenous BCR-related molecules but do express an inducible ERT2- SLP-65 fusion protein which enables examining Ca++influx. All BCRs expressed as IgM effectively mobilized Ca++ without need for an external ligand, indicating autonomous signaling. In contrast, BCRs expressed as IgD did not signal autonomously but required crosslinking with anti-BCR. Thus, only mIgM BCRs naturally transduce a signal in the absence of antigen. To determine the extent that BCR signaling influences clonal activity and in vivoBR, we compared cell size of CLL B cells taken from patients before and after 4 weeks of treatment with the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib (iBTK). Ibrutinib had a strong treatment effect on cell activity, reducing overall cell size in 10/11 patients. A comparison of single cell areas for patients with lower (BR = 0.54%) and higher (BR = 1.42%) BRs showed an overall reduction of the median cell size for both cases. Thus, iBTK treatment leads to an equilibration of the cell size profile among the cases differing in BR, indicating that ibrutinib acts proportionally more potently on more metabolically active CLL B cells. Likewise, these findings are consistent with BCR signaling, transduced through BTK, being responsible for the increased cellular activity of aggressive CLL clones. In conclusion, increased mIgM density and proximity of mIgM to stimulatory receptors is linked to greater metabolic activity clones and increased rate of proliferationin vivo. Conversely, proximity of mIgM to inhibitory receptors has the opposite correlations.Moreover, only mIgM carries out autonomous signaling, providing another biologic trait linking all these features. Thus, our data support a tight, isotype-dependent regulation of BCR signaling and its consequences for CLL B cells. Further understanding these mechanisms should help generate novel therapies to modify the quality of BCR-transduced signaling and thus cell fate. Disclosures Barrientos: Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

scholarly journals A Phase 1 Study of NKX019, a CD19 Chimeric Antigen Receptor Natural Killer (CAR NK) Cell Therapy, in Subjects with B-Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3868-3868
Author(s):  
Michael Dickinson ◽  
Nada Hamad ◽  
Christian E Bryant ◽  
Gautam Borthakur ◽  
Chitra Hosing ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Publicly Traded ◽  
Phase 1 Study ◽  
Advisory Committees ◽  
Car T

Abstract Background: B-cell lineage cancers are a worldwide healthcare burden. Over 500,000 new cases of non-Hodgkin lymphoma (NHL) and 50,000 new cases of acute lymphoblastic leukemia (ALL) are diagnosed world-wide each year (seer.cancer.gov, Smith 2015, Solomon 2017). Despite progress in treatment, many patients diagnosed with this heterogeneous group of cancers still succumb to their disease. Recently approved autologous chimeric antigen receptor (CAR) T cells specific for CD19 have altered the treatment landscape for some patients with relapsed or refractory (R/R) B-cell malignancies, though significant toxicities associated with T-cell expansion and the necessity for bespoke manufacturing have limited their use. Natural killer (NK) cells, part of the innate immune system, efficiently recognize transformed cells and are particularly suited to address limitations of the approved CAR T products (Marcus 2014, Morvan 2016). Lacking a T-cell receptor and the consequent clonal expansion, non-engineered NK cells have been safely administered after lymphodepletion without side effects typically associated with T-cell therapies, such as severe cytokine release syndrome or neurotoxicity (Bachier 2020). Allogeneic NK cell-based therapies allow off-the-shelf use, obviating the necessity to wait for manufacture of autologous T-cell therapies. CD19-directed CAR NK cells have been administered safely, with promising preliminary efficacy (Liu 2020). NKX019 is a cryopreserved product, composed of expanded NK cells engineered to express a humanized CAR against CD19, fused to co-stimulatory (OX40) and signaling (CD3ζ) domains to enhance their intrinsic antitumor activity. NKX019 also expresses a membrane-bound interleukin-15 (IL-15) to serve as an autocrine growth factor and thereby increase NKX019 persistence, with an in vivo half-life of over up to 28 days without systemic IL-2 support. Preclinical characterization has shown that NKX019 cells are 10 times more effective at killing CD19+ target cells than non-engineered NK cells, resulting in greater suppression of xenograft tumor models (Morisot 2020). Further, NKX019, unlike CD19 CAR T cells, retained cytotoxicity even when CD19 antigen density was reduced >50x on target cells. Hence, clinical evaluation of NKX019 is being undertaken in this Phase 1 study in subjects with R/R NHL or ALL. Methods: This is a multicenter, open-label, Phase 1 study of NKX019 (Figure). The study will be conducted in 2 parts: Part 1 (dose finding) to determine the recommended Phase 2 dose (RP2D) of NKX019 separately in adult patients with CAR T naïve R/R NHL or B-ALL, utilizing a "3+3" enrollment schema. Part 2 (dose expansion) will further evaluate safety and tolerability, pharmacokinetics (PK), immunogenicity, pharmacodynamics (PDn), and antitumor activity of NKX019 using RP2D with separate expansion cohorts for patients with ALL as well as different subtypes of NHL, including a cohort of CAR T pretreated large B-cell lymphoma. NKX019 is being manufactured from NK cells obtained from healthy adult donors. The study evaluates two dose levels of NKX019: 3 × 10 8 and 1 × 10 9 viable CAR+ NK cells. NKX019 will be administered on Days 0, 7, and 14 of a 28-day cycle following standard fludarabine/cyclophosphamide lymphodepletion (Table). Up to 5 total cycles may be administered based on response and tolerability assessed at the end of each cycle. The primary endpoint is incidence of adverse events, dose-limiting toxicities, clinically significant laboratory abnormalities, and determination of the RP2D. Secondary endpoints include evaluation of standard cellular PK parameters, PDn, immunogenicity, and antitumor responses. Subjects will be assessed for efficacy using disease-specific criteria: Lugano classification with LYRIC refinement for pseudo-progression (NHL), 2018 International Workshop (IW) criteria (CLL), 6th IW criteria (Waldenström macroglobulinemia [WM]), and National Comprehensive Cancer Version 1.2020 (B-ALL) (Cheson 2006, Cheson 2014, Hallek 2018, Owen 2013, Brown 2020). Enrollment across multiple sites in the US and Australia is expected to start in the second half of 2021. Figure 1 Figure 1. Disclosures Dickinson: Celgene: Research Funding; Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Research Funding; Amgen: Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bryant: Jansen, BMS/Celgene, Skyline Diagnostics: Consultancy; Amgen: Honoraria. Borthakur: Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees. Hosing: Nkarta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shook: Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan: Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rajangam: Nkarta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: SITC: Honoraria; BMS; Karyopharm; Miltenyi: Research Funding; Agios; NGM Biopharmaceuticals; BeiGene: Consultancy. McSweeney: Kite-Gilead: Consultancy; Kite-Gilead, Autolus, Novartis: Research Funding; Kite-Gilead: Honoraria, Speakers Bureau. Hill: Novartis: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria; AstraZenica: Consultancy, Honoraria; Beigene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Pfizer: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria, Research Funding; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Gentenech: Consultancy, Honoraria, Research Funding; Celgene (BMS): Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding.

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.

scholarly journals PARP14 Is a Novel Therapeutic Target in STAT6 mutant Follicular Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2842-2842 ◽  
Author(s):  
Verena Passerini ◽  
Michael Boesl ◽  
Elisabeth Silkenstedt ◽  
Elisa Osterode ◽  
Michael Heide ◽  
...  
Keyword(s):  
Gene Expression ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Board Of Directors ◽  
Molecular Diagnostics ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Wild Type ◽  
Advisory Committees

Abstract The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood. IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23. We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4. RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype. In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. Disclosures Richter: HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

scholarly journals Real-World Treatment of Pediatric Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia Using Tisagenlecleucel That Is out of Specification for Commercial Release

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-44
Author(s):  
Jenna Rossoff ◽  
Christina Baggott ◽  
Snehit Prabhu ◽  
Holly Pacenta ◽  
Christine L Phillips ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Real World ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Single Patient ◽  
Publicly Traded ◽  
Private Company ◽  
Advisory Committees ◽  
Car T

Introduction Chimeric antigen receptor (CAR) T cell therapy has been extremely efficacious in pediatric patients with multiply relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL) with overall remission rates of 81% by three months post-infusion (Maude et al., N Engl J Med, 2018), and achieved FDA approval for this indication. In order for the product to meet the standards of this approval for commercial release, both the leukapheresis and manufactured products must meet a variety of specific requirements, some of which are more stringent than those in these pivotal clinical trials. The Managed Access Program (MAP) provides access to tisagenlecleucel for patients with B-ALL or diffuse large B-cell lymphoma that is out of specification (OOS) for whom repeat leukapheresis is not feasible. Patients may also receive OOS tisagenlecleucel by applying for a single-patient Investigational New Drug (IND). Previous reports have shown no difference in efficacy or toxicity between patients receiving tisagenlecleucel that meets commercial release specifications and those receiving OOS tisagenlecleucel (Grupp, et al., Blood Abstr 614, 2019; Jaglowski, et al., Blood Abstr 627, 2019). This study seeks to evaluate outcomes in pediatric and young adult patients who received tisagenlecleucel via the MAP or a single-patient IND in our Pediatric Real World CAR Consortium (PRWCC). Methods Retrospective data were abstracted from the PRWCC database of patients with relapsed/refractory B-ALL from fifteen different US institutions who received tisagenlecleucel as an FDA-approved therapy outside the context of a clinical trial. Patients whose cellular product was obtained through the MAP (NCT03601442) or with single patient IND approval due having either a cryopreserved leukapheresis product and/or manufactured tisagenlecleucel that did not meet specifications for commercial release were categorized as MAP/IND and those whose product met all release criteria were categorized as standard of care (SOC). Results Among 185 total infused patients in our database, 24 (13%) received tisagenlecleucel either via the MAP (n=14) or a single patient IND (n=10). Baseline patient and disease characteristics were not significantly different for MAP/IND patients versus the SOC cohorts (Table 1). Median duration of follow-up post-CAR T cell infusion for these infused patients was 342.5 days (range 107-780) versus 318 days (range 6-863) for the SOC cohort (p=0.43). Reasons for products being OOS included cell viability <80% (n=17), total nucleated cell count <2x109 in leukapheresis product (n=3), failed interferon gamma release assay (n=2), cryopreserved leukapheresis product collected >9 months prior (n=1), and determination of residual beads >50 beads/3x106cells (n=1). Overall survival at 6- and 12-months was 96% versus 83% and 85% versus 70% for the MAP/IND versus SOC, respectively. Event-free survival at 6- and 12-months was 65% versus 63% and 55% versus 51%, respectively. Probability of continued remission at 6- and 12-months among patients who achieved a complete remission (CR) at day 28 was 79% versus 75% and 66% versus 63% for the MAP/IND versus SOC, respectively (Figure 1). Comparing toxicities between patients in the MAP/IND versus SOC cohorts, cytokine release syndrome (CRS, any grade) occurred in 46% versus 61%, CRS (>grade 3) in 17% versus 19%, immune effector cell-associated neurotoxicity syndrome (ICANS) in 8% versus 22%, and infectious complications in 54% vs. 37%, respectively (p=ns for all). Conclusions In our retrospective cohort evaluating the use of tisagenlecleucel to treat pediatric and young adult patients with relapsed/refractory B-ALL in the real-world setting, neither the efficacy nor safety of tisagenlecleucel seem to be compromised by use of products OOS for commercial release. Larger studies are needed to further delineate specific cut-offs outside of which either efficacy and/or safety may truly be impacted. Disclosures Phillips: Novartis: Membership on an entity's Board of Directors or advisory committees. Stefanski:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Margossian:Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Verneris:Novartis: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Current equity holder in publicly-traded company; Bmogen: Consultancy, Current equity holder in publicly-traded company; Uptodate: Consultancy. Myers:Novartis: Consultancy, Honoraria, Other: ELIANA trial Steering Committee, Speakers Bureau. Brown:Janssen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Qayed:Mesoblast: Consultancy; Novartis: Consultancy. Hermiston:Novartis: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees. Satwani:Mesoblast: Consultancy; Takeda: Consultancy. Curran:Novartis: Consultancy, Research Funding; Celgene: Research Funding; Mesoblast: Consultancy. Mackall:Apricity Health: Consultancy, Current equity holder in private company; Lyell Immunopharma: Consultancy, Current equity holder in private company; BMS: Consultancy; Nektar Therapeutics: Consultancy; Allogene: Current equity holder in publicly-traded company; NeoImmune Tech: Consultancy. Laetsch:Novartis: Consultancy, Research Funding; Bayer: Research Funding; Pfizer: Research Funding; Cellectis: Consultancy.

scholarly journals Tecfidera Modulates Activation of Malignant B-Cells: Correlative Analysis from a Phase 1 Clinical Trial in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3143-3143
Author(s):  
Christina Wu ◽  
Emanuela M. Ghia ◽  
Fitzgerald Lao ◽  
Minya Pu ◽  
Karen Messer ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Phase 1 ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Phase 1 Clinical Trial ◽  
Bcr Signaling

Abstract Tecfidera (Dimethylfumarate, DMF) is an immunomodulatory drug currently approved in the United States for the treatment of patients with multiple sclerosis (MS). DMF formulations have also been utilized for the treatment of patients with psoriasis. The ability of DMF to Inhibit autoreactive T-lymphocytes may also be applicable to malignant T- and B- lymphocytes. Indeed, preclinical studies by multiple groups have highlighted its potential efficacy as a treatment for patients with various lymphoid cancers, though inhibition of STAT, NF-KB, Wnt signaling pathways, and glycolysis. This information provided rationale to conduct a phase 1 clinical trial of DMF as a treatment for patients with relapsed chronic lymphocytic leukemia (CLL). In this study we report the correlative assessment of the initial patients who received DMF. Patients with relapsed or refractory CLL received DMF 120 mg PO BID (based on preclinical modeling with primary CLL cells in vitro and in vivo and 50% lower than the standard DMF dose for patients with MS). Treatment was planned for a short duration of 2 months to evaluate tolerability and effects on CLL cell signaling and tolerability of the drug by patients with CLL. Peripheral blood leukemic cell were isolated and analyzed by RNAseq and phosphoprotein immunoblotting at times just prior to, and 6 hours immediately following drug administration. Consistent with preclinical results (Brennan et al, PLOS one 2015; Selman et al, Science Trans Med 2018), STAT1 phosphorylation at Y701 was decreased 6 hours following the initial dose, compared to high baseline levels. Differential expression analyses for RNAseq of total RNA isolated from peripheral blood CLL cells weere performed using limma models, which compared pretreatment versus post-treatment at a specific time point. Functional annotations of these genes revealed a significant enrichment related to B-cell activation, B-cell receptor (BCR) signaling, B-cell differentiation, and immune signaling generally. Specifically, several known CLL therapy targets including SYK, LYN, and BTK were significantly downregulated. Gene-set-enrichment analysis (GSEA) revealed that the "CLL-BCR gene signature" previously described by Herishanu et al. (Blood 2011) was consistently down-regulated after DMF treatment in samples from different time points (C1D1 post, C1D8 and C2D1) compared to pretreatment samples (FDR q of 0.24, 0.01 and 0.04, respectively). In conclusion, this is the first report of the clinical use of DMF for patients with CLL in which inhibition of leukemic cell activation has been demonstrated. The inhibitory effect of DMF on leukemic cell gene expression, in particular its antagonism of BCR signaling, is valuable information for the development of novel combination strategies for CLL or other B-cell malignancies. Disclosures Kipps: F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Choi:Gilead: Speakers Bureau; Genentech: Speakers Bureau; Rigel: Consultancy; AbbVie, Inc: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau.

scholarly journals A First-in-Human Phase 1 Study of Oral LOXO-338, a Selective BCL2 Inhibitor, in Patients with Advanced Hematologic Malignancies (Trial in Progress)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2424-2424
Author(s):  
Alvaro J. Alencar ◽  
Lindsey E. Roeker ◽  
Marc Hoffmann ◽  
Guru Subramanian Guru Murthy ◽  
Vishalkumar Patel ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Standard Therapy ◽  
Research Funding ◽  
Phase 1 ◽  
Hematologic Malignancies ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Molecule Inhibitor ◽  
Orally Bioavailable

Abstract Background: B-cell lymphoma 2 (BCL2) is a key regulator of apoptosis and provides protection from cell death in many hematological malignancies. The BCL2 inhibitor venetoclax is approved for the treatment of CLL/SLL and acute myeloid leukemia and has activity in other lymphoid malignancies. LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2, designed to achieve selectivity over BCL-xL and thus avoid dose-limiting thrombocytopenia associated with BCL-xL inhibition. In preclinical studies, LOXO-338 showed a favorable pharmacological profile, selectively inhibited BCL2, and was well-tolerated in vivo. LOXO-338 also demonstrated dose-dependent tumor growth inhibition in various murine xenograft models, and showed improved efficacy in combination with pirtobrutinib, a highly selective, non-covalent BTK inhibitor (Brandhuber et al. Cancer Res 2021; 81, 13 Supplement, 1258). Study Design and Methods: LOXO-BCL-20001 is an open-label, multi-center, first-in-human Phase 1 study of oral LOXO-338 in patients with advanced hematologic malignancies who have received standard therapy. The study will be conducted in 2 parts. Part 1 will evaluate LOXO-338 as monotherapy, and will explore different dosing strategies. Part 2 will evaluate LOXO-338 in combination with pirtobrutinib. The dose escalation portion of the study in Part 1 will follow an i3+3 design. Each cycle will be 28 days (4 weeks). Eligible patients include those with CLL/SLL, mantle cell lymphoma (MCL), and Waldenstrӧm macroglobulinemia (WM) who have already received standard therapy. Patients with other B-cell non-Hodgkin lymphomas (NHLs) who failed standard therapy or, in the opinion of the investigator, have no known available options to provide benefit for the patient's condition, are also eligible. Patients must have recovered from prior treatment-related adverse events. Patients with active or suspected Richter transformation, transformed low grade lymphoma, Burkitt or Burkitt-like lymphoma, and multiple myeloma (MM) are eligible in dose-expansion. Key exclusion criteria include history of CNS involvement, stem cell transplant or CAR-T therapy <60 days, concurrent anticancer therapy, and clinically significant cardiovascular disease. The primary objective of Part 1 is to determine the maximum tolerated dose (MTD)/ recommended Phase 2 dose (RP2D) of oral LOXO-338 in patients who were previously treated for CLL/SLL and other B-cell NHLs. Key secondary objectives include determining the safety and tolerability, and pharmacokinetic properties of LOXO-338. Antitumor activity will be evaluated based on overall response rate (ORR), progression-free survival (PFS), time to progression (TTP) and duration of response (DOR) based on disease-specific response criteria per investigator assessment. Key objectives of part 2 are to determine the safety profile and tolerability, PK properties, and anti-tumor activity of LOXO-338 in combination with pirtobrutinib. Disclosures Alencar: Seattle Genetics: Consultancy; Kite Pharma: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Epizyme: Consultancy; Celgene: Consultancy; BeiGene: Consultancy; Amgen: Consultancy. Roeker: AbbVie, AstraZeneca, Janssen, LOXO, Pharmacyclics, TG Therapeutics, Vaniam Group, Verastem: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy, Research Funding; Loxo Oncology: Consultancy; TG Therapeutics: Consultancy; Abbot Laboratories: Current equity holder in publicly-traded company. Hoffmann: TG Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pharmcyclics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria. Guru Murthy: Cancerexpertnow: Honoraria; Guidepoint: Consultancy; Techspert: Consultancy; Qessential: Consultancy; Cardinal Health Inc.: Honoraria; TG therapeutics: Other: Advisory board. Patel: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company. Ku: Loxo Oncology at Lilly: Current Employment, Current holder of stock options in a privately-held company. Pauff: Loxo Oncology at Lilly: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Eyre: Incyte: Consultancy; Loxo Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Gilead/KITE: Honoraria, Other: Travel support for conferences, Research Funding, Speakers Bureau; Secura Bio: Consultancy, Honoraria; Janssen: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel to conferences; AstraZeneca: Honoraria, Research Funding. Jurczak: Celtrion: Research Funding; Celgene: Research Funding; Debbiopharm: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Sandoz: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Epizyme: Research Funding; Incyte: Research Funding; Merck: Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding. OffLabel Disclosure: LOXO-338 is a novel, orally bioavailable small molecule inhibitor of BCL2 for advanced hematologic malignancies.

Diffuse Large B-Cell Lymphoma Patient-Derived Xenograft Models Capture Molecular and Biologic Heterogeneity and Inform Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 817-817
Author(s):  
Bjoern Chapuy ◽  
Honwei Cheng ◽  
Akira Watahiki ◽  
Matthew D Ducar ◽  
Daniel Gusenleitner ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Genetic Alterations ◽  
Advisory Committees ◽  
Large B Cell Lymphoma ◽  
Survival Pathways ◽  
Bcr Signaling ◽  
Pdx Models

Abstract Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease defined by different transcriptional classifications, associated signaling and survival pathways and additional recurrent genetic alterations. In the cell-of-origin (COO) scheme, DLBCLs subtypes share certain features with normal germinal center B-cells (GCB) and activated B-cells (ABC). In comparison to GCB DLBCLs, ABC tumors have increased baseline NFκB activity and more frequent genetic alterations of NFκB pathway components. DLBCLs with shared functional features are also defined by the consensus clustering classification (CCC) which delineates B-cell receptor (BCR), Oxidative Phosphorylation (OxPhos) and Host Response (HR) tumors. BCR DLBCLs have increased reliance on BCR-signaling and survival pathways and aerobic glycolysis. BCR-dependent DLBCLs with high or low baseline NFκB activity (which largely correspond to ABC or GCB tumors, respectively) have distinct SYK-PI3K-dependent survival pathways and shared sensitivity to proximal BCR pathway inhibitors. Although DLBCLs have infrequent inactivating somatic mutations of TP53, these tumors commonly have copy number alterations (CNAs) of TP53 and genes encoding cell cycle pathway components. Given the clinical and molecular heterogeneity of DLBCL, we sought to develop faithful subtype-specific model systems to assess targeted therapies. Fresh tumor biopsies from 27 primary LBCLs were implanted under the renal capsule of immune compromised NSG mice. Nine of 27 tumors were successfully expanded in vivo, serially propagated for > 5 generations and considered stable LBCL PDX models. All models were EBV- and had clonal IgH rearrangements. Morphological and immunohistochemical signatures defined 8 PDX models as DLBCL and 1 as EBV- plasmablastic lymphoma (PBL). All LBCL PDX models were subjected to RNA-Seq and classified with respect to COO and CCC subtypes. Models were also evaluated by whole exome sequencing with a modified bait set which captured coding mutations and selected chromosomal rearrangements. Six of 9 DLBCL PDX models were ABC type. These models exhibited mutations of MYD88 alone or in association with PIM1 or CD79B with other alterations, as reported in primary ABC DLBCLs. The remaining 2 DLBCL PDX models were GCB type, with characteristic alterations of GNA13 and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Of note, BCL2 and MYC translocations are known adverse prognostic features of primary GCB DLBCL. Certain PDX models had additional mutations including B2M, MLL2, TNFAIP3, MEF2B and TP53. Only 25% (2/8) of the DLBCL PDX models harbored inactivating TP53 mutations whereas 75% (6/8) of tumors exhibited CNAs of TP53 or its upstream modifier, CDKN2A. These data are consistent with the reported incidence and type of TP53 pathway alterations in primary DLBCLs and contrast sharply with the near-uniform presence of TP53 mutations in DLBCL cell lines. Using the CCC classification, 6/8 DLBCL PDX models (both GCBs and 4 of 6 ABCs) were defined as BCR-subtype and 2 models as non-BCR type. To assess the utility of the DLBCL PDX models for functional analysis of BCR signaling, we first assessed cell surface immunoglobulin (sIg) expression by flow cytometry. All 6 BCR-type DLBCLs expressed sIgM whereas the 2 non-BCR DLBCL models and the PBL model lacked sIg. Next, we treated viable tumor cell suspensions with a selective SYK inhibitor, entospletinib (GS-9973). SYK inhibition significantly decreased the proliferation of all 6 BCR-type DLBCLs, but had no effect on the non-BCR-type DLBCLs or the PBL PDX. Given the distinctive SYK/PI3K-dependent signaling and survival pathways in DLBCLs with low or high baseline NFκB, we also assessed selective apoptotic pathway readouts in entospletinib-treated PDX cell suspensions. SYK inhibition selectively upregulated the pro-apoptotic BH3 family member, HRK, in BCR-dependent GCB-type DLBCL PDX samples and significantly downregulated the anti-apoptotic BCL2 family member, BCL2A1, in BCR- dependent ABC-type DLBCL PDX tumors, effects consistent with those previously observed in primary DLBCL samples. In summary, we have established and molecularly characterized faithful PDX models of DLBCL and PBL and demonstrated their usefulness in evaluating novel BCR pathway inhibitors. Disclosures Rodig: Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Disease Burden Impacts Outcomes in Pediatric and Young Adult B-Cell Acute Lymphoblastic Leukemia after Commercial Tisagenlecleucel: Results from the Pediatric Real World CAR Consortium (PRWCC)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Liora M. Schultz ◽  
Christina Baggott ◽  
Snehit Prabhu ◽  
Holly Pacenta ◽  
Christine L Phillips ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Board Of Directors ◽  
Real World ◽  
Research Funding ◽  
Disease Burden ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T

Introduction: Chimeric Antigen Receptor (CAR) T cell therapy targeting CD19 has shifted our treatment approach for relapsed and refractory (r/r) pediatric B cell acute lymphoblastic leukemia (ALL). The landmark ELIANA pediatric trial studying tisagenlecleucel, CD19-specific CAR T cells, demonstrated a complete response (CR) rate of 81% in 75 infused patients and 12 month overall survival (OS) and event-free survival (EFS) rates of 76% and 50% respectively. Cytokine release syndrome (CRS) and neurotoxicity rates of 77% and 40% were respectively reported. In August 2017, the FDA approved tisagenlecleucel for B-cell ALL that is refractory or in second or greater relapse in patients up to age 25. With CAR commercialization, institutions deliver tisagenlecleucel without the regulation of a clinical study and practices relating to CAR delivery and reporting remain heterogeneous. Here, we report real world clinical outcomes using commercially available tisagenlecleucel for pediatric r/r B-ALL. Methods and Results: Retrospective data were collected from PRWCC member institutions (n=15) and included 200 patients. This includes 15 (7.5%) patients not infused due to manufacturing failure (n=6), death from disease progression and/or toxicity (n=7), or physician discretion following disease remission from prior therapy(n=2). The remaining 185 patients (92.5%) were infused with tisagenlecleucel, including 87% (161) receiving standard-of-care CAR T cell products meeting manufacturing release criteria and 13% (24) receiving CD19-CAR T cells manufactured by Novartis and provided on the managed access program (NCT03601442; n=14) or with single-patient IND approval (n=10). At time of CAR T cell infusion, median age was 12 years (range 0-26) with 40% females and 60% males. Median duration of follow-up at time of analysis was 11.2 months (range 0.2-28.8). The CR rate at 1 month follow up was 79% (156/198) on an intent-to-treat basis and 85% (156/184) among evaluable infused patients. Of infused patients achieving morphologic CR with available testing, 97% (148/153) were negative for MRD by flow cytometry. Duration of remission at 6 and 12 months among patients who achieved CR was 75% and 63% respectively, with 35% (55/156) of responders experiencing relapse. At time of relapse, 41% (21/51) of evaluable patients had relapse with CD19- disease and 59% (30/51) had continued CD19 expression. OS and EFS rates were 85% and 64% at 6 months and 72% and 51% at 12 months, respectively. CRS and neurotoxicity of any grade were seen in 60% (111/184) and 22% (39/181) of evaluable patients with ≥ grade 3 CRS and neurotoxicity rates of 19% (35/184) and 7% (12/181) respectively. One grade 5 CRS and 1 grade 5 neurotoxicity (intracranial hemorrhage) were reported. Post infusion toxicity management included tocilizumab in 26% (47/184) and systemic steroids in 14% (25/184) of patients. Among 181 infused patients with documented disease burden, 51% (95) had high burden (HB) disease , as defined by >5% bone marrow lymphoblasts, peripheral blood lymphoblasts, CNS3 status or non-CNS extramedullary (EM) site of disease; 22% (40) had low burden (LB) disease, defined by detectable disease not meeting the HB criteria; and 25% (46) had no detectable disease (NDD) at time of last evaluation prior to CAR infusion. The morphologic CR rate was lower at day 28 in HB vs. LB and NDD (74% vs. 98% and 96%) and the OS and EFS were lower among patients with HB at 6 mo [OS; 75% (HB), 94%(LB), 98% (NDD), EFS; 50% (HB), 86% (LB), 75%(NDD), p<0.0001] and 12 mo [OS; 58% (HB), 85% (LB), 95% (NDD), EFS; 34% (HB), 69%(LB), 72%(NDD), p<0.0001]. Multivariate analysis will be presented at the meeting. Conclusions: This retrospective, multi-institutional analysis describes real world outcomes using tisagenlecleucel to treat pediatric r/r B-ALL. Early responses at 1 month and OS and EFS at 6 and 12 months are comparable to reported ELIANA trial outcomes. Safety is demonstrated in this cohort with lower rates or CRS and neurotoxicity, likely related to a lower disease burden cohort. Continued relapse and decrease in OS without evident plateau is seen following 6 months post-infusion warranting expanded follow up. Comparative analysis of outcomes in patient cohorts with varying disease burden demonstrate decreased CR, EFS and OS in patients with high disease burden as compared to patients with lower disease burden or no detectable disease at last evaluation prior to CAR infusion. Disclosures Phillips: Novartis: Membership on an entity's Board of Directors or advisory committees. Stefanski:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Margossian:Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Verneris:Fate Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Membership on an entity's Board of Directors or advisory committees; Bmogen: Consultancy, Current equity holder in publicly-traded company; Uptodate: Consultancy. Myers:Novartis: Consultancy, Honoraria, Other: ELIANA trial Steering Committee, Speakers Bureau. Brown:Jazz: Honoraria; Servier: Honoraria; Janssen: Consultancy; Novartis: Consultancy. Qayed:Novartis: Consultancy; Mesoblast: Consultancy. Hermiston:Novartis: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees. Satwani:Takeda: Consultancy; Mesoblast: Consultancy. Curran:Novartis: Consultancy, Research Funding; Mesoblast: Consultancy; Celgene: Research Funding. Mackall:Lyell Immunopharma: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Apricity Health: Consultancy, Current equity holder in private company; BMS: Consultancy; Allogene: Current equity holder in publicly-traded company. Laetsch:Cellectis: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; Bayer: Consultancy, Research Funding.

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