The U1 Spliceosomal RNA: A Novel Non-Coding Hotspot Driver Mutation Independently Associated with Clinical Outcome in Chronic Lymphocytic Leukemia

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered >80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A>C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A>C point mutation occurring at position 3 of the U1 snRNA gene (g.3A>C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A>C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A>C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A>C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

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Whole Genome Sequencing of B-Cell Neoplasms with t(14;19)(q32;q13) Reveals Different Entities

Blood ◽

10.1182/blood-2021-150891 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3709-3709

Author(s):

Anna Carbó-Meix ◽

Francesca Guijarro ◽

Luojun Wang ◽

Romina Royo ◽

Isabel Granada ◽

...

Keyword(s):

B Cell ◽

Base Pair ◽

Research Funding ◽

Differential Expression Analysis ◽

Lymphocytic Leukemia ◽

Whole Genome ◽

Rna Seq ◽

Molecular Features ◽

Genomic Complexity ◽

Mutational Status

Abstract Introduction: The t(14;19)(q32;q13) is a rare cytogenetic abnormality found in <0.1% of all B-cell neoplasms. The molecular features of this translocation are not well characterized. IGH-BCL3 rearrangement has been found in some tumors identified as "atypical" chronic lymphocytic leukemia (CLL) with aggressive clinical evolution. This translocation has also been observed in other B-cell neoplasms without clear evidence of the target gene. The mechanisms generating this translocation, the genomic profile of alterations of these cases, and whether different molecular features may be associated with specific entities are not known. Aim: To elucidate the genomic features of B-cell neoplasms carrying the t(14;19) and their relationship to pathological characteristics of the tumors. Materials and methods: We sequenced the whole-genome (WGS) of 13 cases in which the t(14;19) had been identified by conventional cytogenetics and/or FISH using a BCL3 break-apart probe. In six of these cases we performed RNA-seq. Pathological and clinical revision was conducted in all cases, 8 of them with tissue biopsies. Results: The breakpoints of the t(14;19) were characterized at base-pair resolution using WGS. All breakpoints in chr14 were found within any of the class switch recombination (CSR) regions suggesting an aberrant CSR as the mechanism causing this alteration. The breakpoints on chr19 were found upstream (13 kb) the 5' untranslated region (UTR) of BCL3 in 8/13 (61.5%) cases. One additional case had the breakpoint further upstream (49 kb) of BCL3 truncating CEACAM16. The four remaining cases had breakpoints downstream of BCL3; two cases within CBLC, one in BCAM, and one after NECTIN2. Of note, the further upstream BCL3 case and the downstream BCL3 cases had mutated IGHV, while all upstream BCL3 cases had unmutated IGHV. Based on RNA-seq data, all upstream BCL3 cases (n=5) showed an upregulation of BCL3, while one downstream case with RNA-seq available showed upregulation of NECTIN2 and low levels of BCL3. The pathology review identified the four downstream BCL3 cases as marginal zone lymphomas whereas the cases with breakpoints upstream BCL3 (n=3 with tissue available) and the case further upstream BCL3 were classified as "atypical" CLL. We next characterized the genomic landscape of these tumors based on the breakpoint on chr19 (upstream and downstream BCL3). The analysis of the WGS showed a lower number of mutations, copy number alterations (CNA), and structural variants (SV) in the upstream BCL3 group compared to the downstream BCL3 cases (mean of 2429.5 vs 6271.7 somatic mutations, 3.1 vs 11.7 CNA, and 4.4 vs 18 SV, respectively). In terms of specific driver mutations, the downstream BCL3 group carried mutations in genes previously described in MZL, such as KMT2D, NOTCH2, or KLF2 found in two cases. All but one case with the breakpoint upstream BCL3 carried trisomy 12 (tri12), which was absent in all cases with a downstream breakpoint. Finally, we performed a differential expression analysis between 5 atypical CLL cases with BCL3 rearrangements vs 4 CLL without t(14;19) [all unmutated IGHV]. This analysis showed 578 genes upregulated and 720 genes downregulated in the BCL3-rearranged cases (q <0.05), including remarkable differences in the expression of previously described CLL hallmark genes, such as upregulation of EBF1 and downregulation of LEF1, FMOD, ADTRP, CLNK, IGSF3, TCF4. An analysis of the RNA-seq data of 294 CLL cases lacking the t(14;19) (Puente et al., Nature 2015) indicated that this transcriptional program was not related to IGHV mutational status nor to the presence of tri12. Nonetheless, we identified a small set of tri12 mutated IGHV CLL lacking the t(14;19) with a similar modulation of the expression of the above hallmark genes. Conclusions: We have characterized the breakpoints of the t(14;19) at base-pair resolution and evidenced marked molecular and pathological differences of the tumors according to the location of the breakpoint. Tumors carrying the breakpoint downstream BCL3 exhibit a higher genomic complexity, driver alterations, and pathological features corresponding to MZL. Contrarily, tumors with the breakpoint upstream of BCL3 upregulate BCL3 and display lower genomic complexity as well as CLL-like features. Nonetheless, these cases have a different gene expression profile compared to conventional CLL characterized by LEF1 downregulation and EBF1 overexpression. Disclosures Navarro: Nocartis: Honoraria; Roche: Honoraria; EUSA: Consultancy, Research Funding; Pharma: Consultancy; GILEAD: Research Funding; Pharma: Research Funding.

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Secreted Factors Determine Resistance to Idelalisib in Marginal Zone Lymphoma Models of Resistance

Blood ◽

10.1182/blood-2019-124299 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2569-2569

Author(s):

Alberto J Arribas ◽

Sara Napoli ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Alessandra Di Veroli ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Institutional Research ◽

Rna Seq ◽

Scientific Advisory Board ◽

Scientific Advisory ◽

Erbb Signaling ◽

Travel Grant

Background . PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines. Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA. Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities. The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening. In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line. Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going. Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P <0.05). Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches. Disclosures Stathis: PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.

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Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽

10.1182/blood-2021-149442 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 248-248

Author(s):

Alice Bonato ◽

Riccardo Bomben ◽

Supriya Chakraborty ◽

Giulia Felician ◽

Claudio Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Pi3k Inhibitor ◽

Leukemia Cells ◽

Wild Type ◽

Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

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SYK Inhibition Disrupts the Cross-Talk Between B-Cell Activation Factor (BAFF) and B-Cell Receptor (BCR) and Thereby Antagonizes Mcl-1 in Chronic Lymphocytic Leukemia (CLL) B-Cells

Blood ◽

10.1182/blood.v128.22.303.303 ◽

2016 ◽

Vol 128 (22) ◽

pp. 303-303

Author(s):

Cody Paiva ◽

Taylor Rowland ◽

Olga Danilova ◽

Bhargava Sreekantham ◽

Stephen E Spurgeon ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Cell Survival ◽

Research Funding ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Mutational Status ◽

Pathway Gene ◽

Bcr Signaling

Abstract Although small molecule inhibitors of BCR-associated kinases (BCRi) revolutionized therapy in CLL, they provide incomplete responses. Soluble mediators emanating from the tumor microenvironment perpetrate CLL cell survival and may account for resistance to BCRi. Tumor necrosis factor receptor superfamily ligands BAFF and APRIL induce NFκB, which in turn upregulates pro-survival Bcl-2 family proteins and thereby drives anti-apoptotic responses.The exact roles of the individual NFκB pathways, as well as the implications of targeting BCR in context of BAFF signaling in CLL remain understudied. We explored the mechanistic underpinnings of CLL cell survival in response to BAFF signaling, uncovering the functional significance of the BCR-associated kinases and Bcl-2 family proteins in this setting. Peripheral blood mononuclear cells were isolated from patients with CLL. We established a novel BAFF-expressing stromal co-culture model and referenced it to control, CD40L-expressing stroma and soluble BAFF. We employed inhibitors of Bruton tyrosine kinase (BTK, ibrutinib), phosphoinositide-3 kinase (PI3K, idelalisib) and spleen tyrosine kinase (SYK, entospletinib) and measured CLL cell apoptosis, migration, NFκB activity, protein and mRNA expression by flow cytometry, immunoblotting, ELISA, RT-PCR and immunocytochemistry. CLL cells co-cultured with BAFF-expressing stroma were resistant to spontaneous apoptosis (12.3±3.2% after 24 h, vs 34.8±6.2% off stroma) and chemotherapy agents (bendamustine, fludarabine). Gene expression profiling exposed the NFκB pathway gene targets as the most significantly upregulated upon BAFF stimulation (p<0.0001). We and others have shown that CD40L-expressing stroma induces canonical and non-canonical NFκB in CLL. By contrast, while BAFF led to strong activation of the non-canonical NFκB with processing of p100 (to p52) by 4 h and a 5-fold increase in p52 DNA-binding activity by 24 h, canonical NFκB (RelA) activation was less pronounced. BAFF predominantly induced Mcl-1, compared to CD40L which strongly upregulated Bcl-X. BCR is a major driver of canonical NFκB signaling in CLL. Thus, we studied whether BAFF co-opted BCR signaling in CLL. BAFF induced rapid (15 min) phosphorylation of the proximal BCR kinases SYKand LYN, sustained for up to 4 h, as well as ERK, in CLL cells. AKT activation occurred late (>2 h), suggesting that BAFF induced AKT independent of BCR. BAFF-mediated BCR activation did not correlate with IGHV mutational status. Like IgM, BAFF induced CLL cell chemotaxis. SYK inhibition effectively antagonized survival and chemotaxis of BAFF-stimulated CLL cells. By contrast, targeting BTK or PI3K was less effective. All BCRi's fully blocked canonical NFκB activation in BAFF-stimulated CLL cells (suggesting its dependence on BCR signaling), but none inhibited the non-canonical pathway. We found that entospletinib, but not other BCRi's, decreased Mcl-1 expression in CLL cells co-cultured with BAFF-expressing stroma. Unlike in IgM-stimulated cells, entospletinib did not promote Mcl-1 protein degradation. By contrast,, targeting SYK in BAFF-stimulated cells abrogated BAFF-mediated upregulation of pSTAT3, a transcription factor which regulates Mcl-1. This was accompanied by a decrease in Mcl-1 transcript, an effect mimicked by ruxolitinib, a JAK/STAT inhibitor. BAFF receptor signals via the TRAF3/NIK/IKK1 axis to induce non-canonical NFκB activation in neoplastic B-cells. We supposed that NIK (NFκB-inducing kinase) or IKK1 could be directly responsible for SYK activation by BAFF. Indeed, genetic knockdown of NIK resulted in decreased SYK activation, whereas IP experiments demonstrated that NIK directly complexed with SYK in BAFF-stimulated neoplastic B-cells, confirming NIK role in activation of BCR signaling. Thus, BAFF-mediated induction of BCR-associated kinases and Mcl-1 contributes to CLL cell survival. SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and BCR, thereby disrupting the pro-survival microenvironment signaling in CLL. Disclosures Spurgeon: Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Danilov:Prime Oncology: Honoraria; Dava Oncology: Honoraria; ImmunoGen: Consultancy; GIlead Sciences: Research Funding; Takeda: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Consultancy.

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A Novel Spirocyclic Dimer (36-286) Targeting the NF-Kappa B Pathway Displays Potent Anti-Tumor Properties in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2021-152632 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1186-1186

Author(s):

Alexandria P Eiken ◽

Audrey L Smith ◽

Sarbjit Singh ◽

Sandeep Rana ◽

Sunandini Sharma ◽

...

Keyword(s):

B Cells ◽

Cell Growth ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Protein Translation ◽

Lymphocytic Leukemia ◽

Rna Seq ◽

Resistant Cells

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is an incurable, heterogenetic disease dependent on B cell receptor (BCR) signaling with subsequent nuclear factor-kappa B (NF-κB) activation resulting in the evasion of apoptosis and enhanced malignant B cell growth. Targeted therapies such as ibrutinib (IBR; BTK inhibitor) and venetoclax (VEN; BCL2 antagonist) have revolutionized the management of CLL, however ~20% of patients relapse, signifying the urgent need for novel therapeutics for CLL patients especially those with refractory/relapse (ref/rel) disease. Additionally, various tumor microenvironment (TME) stimuli fuel CLL growth and contribute to drug resistance through the activation of numerous signaling pathways (BCR, CD40R, TLR, BAFFR) and consequential sustained NF-κB activation. Currently, there are no FDA approved drugs that effectively target the NF-κB protein family. Herein we introduce 36-286 (N3), a novel spirocyclic dimer which displays NF-κB inhibitory activity and elicits potent anti-leukemic properties. N3 is a dimer of a spirocyclic α-methylene-γ-butyrolactone analog that covalently binds to surface exposed cysteine residues on NF-κB proteins (IKKβ and P65) (Rana S et al, 2016). Our study aims to investigate N3's mode of action (MOA) and to establish its anti-leukemic effects in CLL including drug-resistant disease, thereby introducing a novel therapeutic option for rel/ref disease. Methods: Cell growth via MTS proliferation assay was determined following treatment with N3 (0.125 - 2 μM) in a panel of malignant B cell lines [CLL (HG3, MEC1, OSUCLL), diffuse large B cell lymphoma (Pfeiffer, RC, RIVA), mantle cell lymphoma (Jeko1)], and in patient derived CLL cells stimulated with CpG ODN 2006 (CpG; 3.2 μM). Viability testing of normal B cells isolated from healthy donors was conducted following N3 treatment. Anti-tumor properties of N3 (1 - 2 μM; 4h) in the HG3 and OSUCLL cell lines were further confirmed under conditions mimicking different TME stimuli such as α-IgM (10 μg/mL), CD40L (100 ng/mL), BAFF (50 ng/mL) or CpG (3.2 μM). Protein expression of oncogenic MYC, select NF-κB pathway proteins (IKKα, IKKβ, P65, IκBα, RelB) and the anti-apoptotic protein MCL1 was determined following treatment with N3 (0.25 - 2 μM; 4h) by immunoblot (IB). Next, we induced IBR resistance in HG3 cells by prolonged exposure to increasing IBR concentrations (~10-15 fold its IC 50 in parental cells). Cell proliferation via MTS was determined following treatment with N3 on these resistant cells. To gain insight on the potential MOA of N3 in CLL, we adapted a proteomics-based approach (TMT labeled mass spectrometry) and conducted RNA-seq in OSUCLL cells treated with N3 (1 - 2 μM) for up to 24 h. Subsequent pathway analysis was performed to identify the top factors modulated by N3. Results: N3 showed remarkable efficacy (IC 50 < 0.6 μM) across all the malignant B cell lines evaluated while sparing normal B cells. In CpG stimulated primary CLL, N3 resulted in marked anti-leukemic effects (0.125 μM) comparable to IBR (1 μM). N3 induced cell apoptosis in CLL cell lines in a dose-dependent manner with marked PARP cleavage. Furthermore, our IB analyses of N3 treated CLL cell lines showed reduced levels of NF-κB pathway proteins, MYC and MCL1. Notably, N3 was effective in reducing levels of the above-mentioned proteins in the presence of the various TME stimuli. Strikingly, N3 maintained its cytotoxic effects in ibrutinib resistant HG3 cells. Studies to confirm N3's cytotoxicity in VEN resistant CLL cells are ongoing. Top ten pathways from both proteomics and RNA-seq analyses revealed an upregulation of the unfolded protein response (UPR) and inhibition of cap-dependent protein translation. IB analyses of select factors related to UPR (CHOP, XBP1, PERK, IRE1) and protein translation (eIF2α, 4E-BP1, PDCD4) in N3 treated CLL cells validated our omics' findings. Efforts to identify the proteome wide direct targets of N3 in CLL cells are currently underway. Conclusion: N3 is a novel pre-therapeutic lead that targets multiple survival and proliferation pathways through the inhibition of NF-κB activity and upregulation of UPR. We show that its highly cytotoxic in tumor B cells while sparing normal B cells. Moreover, N3 sustained its anti-tumor properties under different TME stimuli and in IBR resistant cells, indicating the potential use of this compound in rel/ref patients following evaluation in murine CLL models. Disclosures No relevant conflicts of interest to declare.

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MYD88 Triggered SYK Activation Promotes BCR Cross-Talk, and Identifies SYK As a Novel Therapeutic Target of Mutated MYD88 Signaling

Blood ◽

10.1182/blood-2018-99-117372 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4116-4116

Author(s):

Manit Munshi ◽

Jiaji Chen ◽

Xia Liu ◽

Lian Xu ◽

Nickolas Tsakmaklis ◽

...

Keyword(s):

Tumor Cell ◽

B Cell ◽

Cell Lines ◽

Cross Talk ◽

Research Funding ◽

Wild Type ◽

Over Expression ◽

Activating Mutations ◽

Bcr Signaling ◽

Myd88 Signaling

Abstract Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. Disclosures Castillo: Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

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Integrative Transcriptome and Quantitative Proteome Analyses Identify METTL3 As a Key Regulator for Aberrant RNA Processing in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-139841 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12

Author(s):

Yiming Wu ◽

Meiling Jin ◽

Kevyn Hart ◽

Aijun Liao ◽

Stacey M. Fernandes ◽

...

Keyword(s):

B Cells ◽

Cell Lines ◽

Research Funding ◽

Stop Codon ◽

Protein Translation ◽

Rna Metabolism ◽

Mrna Processing ◽

Lymphocytic Leukemia ◽

Rna Seq ◽

Healthy Donors

Aberrant mRNA processing is known to drive the pathogenesis of chronic lymphocytic leukemia (CLL). Recurrent gene mutations in the RNA splicing factor SF3B1 and widespread RNA intronic polyadenylation impact genome-wide gene expression and inactivate tumor suppressors, respectively. Nevertheless, how mRNA processing is regulated and exerts its function in CLL remain elusive. To comprehensively characterize the role of mRNA processing in CLL, we performed RNA sequencing (RNA-seq) and Tandem Mass Tag (TMT) proteomics using normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=22). We detected 328 proteins differentially expressed between normal and CLL B cells (|Log2FC|>0.58, q<0.05). Gene set enrichment analysis (GSEA) revealed that proteins involved in RNA metabolism (transcription, splicing, modification, 3'end processing, nuclear export, decay) were upregulated in CLL, while those impacting translation were downregulated. These findings were validated by immunoblotting in an independent set of samples (n=10). However, we observed no significant gene expression changes of RNA metabolism at the transcript level, indicating that regulation of these proteins occurred post-transcriptionally. Since N6-methyladenosine (m6A) is the most abundant RNA internal modification and has emerged as a key regulator for RNA metabolism, we sought to determine whether m6A is dysregulated in CLL cells. With an m6A dotblot assay and HPLC-MS, we consistently detected increased level of m6A in mRNA from CLL cells compared with normal B cells. As one of the most upregulated proteins in CLL, METTL3 writes m6A and promotes translation efficiency through its writer and reader functions, respectively. When we knocked down (KD) METTL3 in CLL cell lines (HG3, MEC1) as well as in primary CLL cells, we observed significant cell death and growth disadvantage in CLL compared to control cells, highlighting METTL3 is essential for CLL survival. We next examined whether KD of METTL3 affects m6A and RNA translation using m6A dotblot and O-propargyl-puromycin run on assays. Loss of METTL3 had subtle impact on m6A levels but it significantly decreased protein translation (t test, p<0.01) in all the cell lines tested (HG3, MEC1, JeKo-1, Mino). To define the target protein that METTL3 affects, we performed an integrated Ribosome profiling and RNA-seq analysis using HG3 and Mino cells with or without METTL3. At both transcriptome and translatome levels, loss of METTL3 significantly decreased genes enriched in the mTORC1 pathway, which has an essential role in translation (Metascape, hypergeometric test, q<0.05). Furthermore, it also decreased the translation efficiencies of genes involved in mRNA processing, DNA synthesis, and cell cycle pathways. This observation suggests that upregulation of METTL3 in CLL cells may regulate protein translation of the RNA metabolism pathway. Since m6A at the stop codon region is critical for METTL3 regulating protein translation, we performed MAZTER sequencing to determine m6A modification sites in normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=11). We identified 214 genes with significant differential m6A modification at the stop codon region (delta cleavage efficiency>0.1, Wilcoxon rank-sum test, p<0.1, within DRACA motif) between normal and CLL B cells. These genes were highly enriched for mRNA processing (Metascape, q=0.017), supporting our notion that METTL3 may modulate protein expression of mRNA processing genes by recognizing m6A modification via its reader function in CLL. Consistent with its role in regulating protein expression, we detected downregulation of splicing factors (SF3A1, SF3A2, SF3B1, U2AF1) in various METTL3 KD cell lines (HG3, MEC1, JeKo-1, Mino) at only protein level but not transcription level. These data link METTL3 upregulation with RNA metabolism protein enrichment in CLL. Altogether, our integrated analysis uncovered a novel regulatory axis of METTL3 in CLL biology. We demonstrated that CLL cells have an increased m6A modification and upregulation of METTL3 at the protein level, resulting in translation of RNA metabolism related genes through its reader function by the recognition of m6A modification. Our results collectively suggest METTL3 as a central regulator for mRNA processing in CLL and provide a rationale for targeting METTL3 in this disease. Disclosures Brown: Janssen, Teva: Speakers Bureau; Gilead, Loxo, Sun, Verastem: Research Funding; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy. Rosen:Seattle Genetics: Consultancy; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau. Siddiqi:TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Oncternal: Research Funding; BeiGene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau.

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BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia and Lymphoma

Blood ◽

10.1182/blood.v128.22.2767.2767 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2767-2767

Author(s):

Deborah M Stephens ◽

Kyle A. Beckwith ◽

Priscilla Do ◽

Carolyn Cheney ◽

Xiaokui Mo ◽

...

Keyword(s):

B Cells ◽

Cell Viability ◽

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Annexin V ◽

Lymphocytic Leukemia ◽

Advisory Committees

Abstract Background Targeting new antigens in chronic lymphocytic leukemia (CLL) and lymphoma may increase flexibility in the clinic and help circumvent resistance. The tetraspanin CD37 domain mediates transduction of survival and apoptotic signals (Lapalombella et al.,Cancer Cell, 2014), and has been clinically validated by recent trials of otlertuzumab (TRU-016) in CLL and Non-Hodgkin Lymphoma . Ligation of CD37 by this reagent simultaneously induced pro-apoptotic signaling and inhibited pro-survival signaling of phosphoinositide 3-kinase δ (PI3Kδ), which introduces a unique opportunity to use combination strategies employing activation of CD37 and inhibition of PI3Kδ. A new agent BI 836826 is an Fc-engineered anti-CD37 IgG1 that displays improved effector activities as well as crosslinker-independent direct cytotoxicity. We have evaluated the efficacy of BI 836826 combined with the PI3Kδ-selective inhibitor idelalisib in diffuse large B-cell lymphoma (DLBCL) cell lines and primary human CLL B-cells in the University and then by industry to validate the synergistic finding initially reported. Methods Cell viability assays usedCellTiterGlo to measure inhibition of antibody, isotype control, idelalisib or a combination of antibody and compound over 72h in culture. The cell viability of vehicle is measured at the time of dosing (T0) and after seventy-two hours (T72). A GI reading of 0% represents no growth inhibition, GI 100% represents complete growth inhibition, and a GI 200% represents complete death of all cells in the culture well. Annexin V-FITC and propidium iodide measure by flow cytometry was used to assess enhanced killing of primary CLL cells, with incubation of BI 836826 (0.1 µg/mL) and/or idelalisib (1 µM) at 37°C for 24 hours. Trastuzumab included as a non-specific IgG1 control. Data was reported as percentage of viable cells (Annexin V negative, PI negative) normalized to untreated control. Results DLBCL cell lines were variably sensitive to single agent BI 836826. In most of the cell lines tested, the cell viability was inhibited by 40%-50% with BI 836826 in the concentration range of 1-1000 ng/mL (Figure 1A). A synergistic effect was noted in several DLBCL cell lines when BI 836826 was combined with idelalisib. When the maximal effect of BI 836826 was greater than isotype control (GI% > 12, dotted line) and the effect of idelalisib showed a GI50 < 1uM, 3/5 cell lines showed synergy in combination (red dot, Figure 1B). A shift in the EC50of idelalisib can be seen with the addition of increasing amounts of BI 836826 (Figure 1C). In primary CLL B-cell cultures, 1 µM idelalisib displayed weak single agent activity following 24-hour incubation. The cytotoxicity of BI 836826 at 0.1 µg/mL was more variable, although treatment of samples from most CLL patients resulted in 20-50% B-cell death. The combination of these 2 agents resulted in enhanced cytotoxic activity (Figure 2A), and this effect was not attenuated by the presence of del(17)(p13.1), as there was no significant difference in cytotoxicity against these cells compared to those with lower risk cytogenetics (Figure 2B,C). Additionally, the combination was beneficial in CLL B-cells isolated from patients who were refractory to ibrutinib (Figure 2D). Conclusions This collaborative industry and academic endeavor with cross validation of initial mechanistic studies of synergy between CD37 and idelalisib demonstrates that addition of idelalisib to BI 836826 augments cytotoxicity against DLBCL cell lines and primary human CLL B-cells in an additive-to-synergistic manner. In addition, it maintains efficacy against CLL B-cells with del(17)(p13.1) and those from ibrutinib-refractory patients. Further exploration of this therapeutic strategy in clinical trials is strongly warranted. Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Grosmaire:Gilead: Employment. Jones:Gilead: Employment. DiPaolo:Gilead: Employment. Tannheimer:Gilead Sciences: Employment. Heider:4Boehringer Ingelheim RCV: Employment.

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A Novel HCK Inhibitor Kin-8193 Blocks BTK Activity in BTKCys481 Mutated Ibrutinib Resistant B-Cell Lymphomas Driven By Mutated MYD88

Blood ◽

10.1182/blood-2018-99-119171 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 40-40

Author(s):

Guang Yang ◽

Jinhua Wang ◽

Li Tan ◽

Xia Liu ◽

Manit Munshi ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Research Funding ◽

Kinase Inhibitor ◽

Target Engagement ◽

Wild Type ◽

B Cell Lymphomas ◽

Oral Dosing ◽

Activating Mutations ◽

Ibrutinib Resistance

Abstract Activating mutations in MYD88 alone or in coordination with BCR activating mutations transactivate Bruton's tyrosine kinase (BTK) in WM and ABC DLBCL cells (Yang et al, Blood 2013; Wilson WH et al, Nat Med 2015; Phelan JD, Nature 2018). Ibrutinib is a covalent inhibitor that binds to BTKCys481 and is active in MYD88 mutated WM and ABC-DLBCL (Treon et al, NEJM 2015; Wilson et al, Nat Med, 2015). Acquired resistance to ibrutinib is increasingly being recognized in WM, as well as other B-cell malignancies due to somatic mutations at BTKCys481 that abrogate BTK-ibrutinib binding (Xu et al, Blood 2017). BTKCys481 mutations are usually sub-clonal, but can drive ibrutinib resistance and protect BTK wild-type clones through a paracrine mechanism involving activation of ERK1/2 (Chen et al, Blood 2018). Hematopoietic cell kinase (HCK) which is aberrantly upregulated and transactivated by mutated MYD88, and triggers multiple pro-survival signaling cascades that include activation of BTK, as well as PI3K/AKT and MAPK/ERK1/2 (Yang G et al, Blood 2016). We therefore examined if inhibition of HCK could abrogate BTK activity and overcome ibrutinib resistance driven by BTKCys481 mutations in MYD88 mutated B-cell lymphomas. We performed a screen of 220 clinical and preclinical kinase inhibitors to identify compounds with potent HCK inhibition. Over 100 analogs of three series of promising compounds with HCK activity were synthesized and triaged. Target deconvolution was performed to clarify selectivity, and other important kinase targets. These efforts led to the selection of a lead compound identified as a type-II kinase inhibitor, KIN-8193, with a molecular weight around 600. Single-digit nanomolar (nM) biochemical and double-digit nM cellular potency was demonstrated, with high selectivity (S-score 0.07) in line with that observed with ibrutinib (S-score of 0.03). Live-cell target engagement for HCK was confirmed by competitive ATP-biotin binding assay. DMPK and PK studies showed very high levels of mouse, rat, and human microsomal stability (42.4, 60.2 and 79.3 minutes respectively), and oral bioavailability in mice (48%) and rats (79%). Cmax reached 2.0 µM, while T1/2 was 26.8 hours with 25 mg/kg single oral dosing in rats. Rats toxicology studies showed excellent tolerability in 28-days repeated oral dosing with 25 mg/kg/biw dosing schedule. No relevant inhibition was observed against a panel of 100 other receptor targets, including hERG. AMES was negative up to 100 uM, and Cyp inhibition studies showed acceptable inhibition up to 10 uM. KIN-8193 potently inhibited the phosphorylation of HCK(p-Y411) and its downstream target BTK(p-Y223) in both BTK wild-type and ibrutinib-resistant BTKCys481 mutated WM and ABC DLBCL cell lines driven by activating MYD88 mutations, and primary WM cells (Fig 1A). Target engagement studies showed HCK but not BTK direct binding. In vivo pharmacodynamic (PD) studies using luciferized BCWM.1 cells WM xenograft mouse model showed potent dose-dependent inhibition of HCK(p-Y411) and BTK(p-Y223) at 6 and 24 hours following single dose administration of KIN-8193. Importantly, KIN-8193 showed selective cytotoxicity against MYD88 mutated BTK wild-type and BTKCys481 mutated, ibrutinib-resistant WM and ABC DLBCL cell lines, and primary WM cells, but had no impact on healthy donor B- and T-cells at pharmacologically achievable levels (Fig. 1B). We therefore describe a novel, highly selective and potent HCK inhibitor that is well tolerated in long-term rat toxicology studies and shows selective killing of MYD88 mutated WM and ABC DLBCL cells. Inhibition of HCK by KIN-8193 blocks downstream wild-type BTK and Cys481 mutated BTK activity, and overcomes ibrutinib resistance induced by BTKCys481 mutations. Disclosures Hunter: Pharmacyclics: Consultancy. Castillo:Genentech: Consultancy; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Millennium: Research Funding. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

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Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-145956 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 615-615

Author(s):

Yuxuan Liu ◽

Lucille Stuani ◽

Dorra Jedoui ◽

Milton Merchant ◽

Astraea Jager ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Bcr Signaling ◽

Proliferation In Vitro

Abstract Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

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