Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) and CLL with del17p. Mechanistically, ibrutinib interferes with ...B-cell receptor (BCR) signaling as well as multiple CLL cell-to-microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted.
We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole-exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 to 5 μmol/L of ibrutinib
and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted
in HG3, PGA1, and PG-EBV cell lines and measured BCR signaling and ibrutinib responses.
CLL samples demonstrated a surprisingly wide range of
sensitivities to ibrutinib, with IC
values ranging from 0.4 to 9.7 μmol/L. Unmutated IGVH status, elevated ZAP70 expression, and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/
-mutated status. A validation sample of 15 CLL carrying
mutations, of which 13 carried both del17p and a
mutation, confirmed substantially less sensitivity to ibrutinib-induced apoptosis.
This study identifies that CLL harboring del17p/
-mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/
wild-type cells.
.
The recent discovery of genes mutated in chronic lymphocytic leukemia (CLL) has stimulated new research into the role of these genes in CLL pathogenesis. CLL cases carry approximately 5-20 mutated ...genes per exome, a lower number than detected in many human tumors. Of the recurrently mutated genes in CLL, all are mutated in 10% or less of patients when assayed in unselected CLL cohorts at diagnosis. Mutations in TP53 are of major clinical relevance, are often associated with del17p and gain in frequency over time. TP53 mutated and associated del17p states substantially lower response rates, remission duration, and survival in CLL. Mutations in NOTCH1 and SF3B1 are recurrent, often associated with progressive CLL that is also IgVH unmutated and ZAP70-positive and are under investigation as targets for novel therapies and as factors influencing CLL outcome. There are an estimated 20-50 additional mutated genes with frequencies of 1%-5% in CLL; more work is needed to identify these and to study their significance. Finally, of the major biological aberration categories influencing CLL as a disease, gene mutations will need to be placed into context with regard to their ultimate role and importance. Such calibrated appreciation necessitates studies incorporating multiple CLL driver aberrations into biological and clinical analyses.
Introduction: Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p (all lines). Overall response rates to ibrutinib in treatment ...naïve or relapsed CLL are high and most patients experience prolonged remission durations, although cases of acquired ibrutinib resistance have been reported.
Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment cell interactions. Weakening of these interactions underlies some of the mechanisms by which ibrutinib is working in vivo. However, comparatively little is known about CLL cell-intrinsic determinants of apoptotic responses induced by ibrutinib. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance and ultimately clinical outcome are warranted.
Methods: We first characterized 50 paired CLL samples procured before and after standard CLL therapies (predominantly chemo immunotherapies) for IGVH status, ZAP70 positivity, CD38 expression, TP53 mutations and FISH and analyzed the relapsed samples by whole exome sequencing (WES). Preliminary ibrutinib treatment studies using CD40-expressing fibroblasts or IL-4 or both in CLL co-culture resulted in low ibrutinib activity, precluding measurements of cell-intrinsic response determinants. Therefore, to assay ibrutinib-mediated CLL cell kill, CLL samples were purified using negative selection and incubated in RPMI medium supplemented with 10% FCS for 72 hours with ibrutinib at varying doses (0.25 µM - 5 µM). Cell death and viability was measured using AnnexinV-PI staining and flow cytometry. IC50 values were calculated using the program XLfit.
Results: CLL cells demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.37 µM - 9.69 µM in pre-treatment samples and 0.56 µM - >10 µM in post treatment samples (mean IC50 values for pre-treatment and relapsed samples were 2.7 µM and 3.7 µM, respectively). Multiple cell-intrinsic determinants of IC50 values to ibrutinib were identified; CLL samples from IGVH unmutated samples were more sensitive than samples from IGVH mutated samples (mean IC50 values for pre-treatment samples were (IGVH -UM) 2.1 µM and (IGVH -M) 3.5 µM (p=0.01), and relapsed samples of (IGVH -UM) 2.8 µM and (IGVH -M) 4.0 µM (p=0.03), respectively, consistent with a greater dependency/signaling capacity of the BCR in IGVH-UM CLL. Similar findings were derived for ZAP70 status, with CLL samples with <20% ZAP70 positivity by FACS being less sensitivity to ibrutinib than ZAP70 positive samples (mean IC50 values for relapsed CLL samples were 4.1 µM and 2.8 µM, respectively; p=0.03). Trisomy 12 was also associated with sensitivity to ibrutinib (mean IC50 values for trisomy 12 positive rCLL samples were 2.8 µM and 4.1 µM, respectively; p=0.03).
IC50 results for paired samples demonstrated a strong concordance within paired samples (Pearson correlation coefficient 0.66). However, ~10% of CLL samples were substantially more resistant to ibrutinib at relapse when compared with their paired diagnosis samples. Of these 5 samples, three had acquired mutations in TP53 following prior chemotherapy; a hypothesis-generating finding that acquired TP53 mutations reduce ibrutinib sensitivities. An expansion cohort of 7 additional ex vivo ibrutinib-treated TP53-mutated CLL samples confirmed the novel and clinically relevant finding that TP53 mutated CLL cells are substantially less sensitive to ibrutinib than TP53 wild type cells (mean IC50 values of 6.1 µM and 2.7 µM, respectively; p<0.001)
Conclusion: Ibrutinib ex-vivo treatment of 50 paired CLL samples procured before and after standard CLL therapies identified cell-intrinsic determinants of ibrutinib sensitivity, including IGVH unmutated CLL and trisomy 12 and implicates prior chemotherapy-selected mutations in TP53 as an intrinsic cause of reduced response. Expansion data identified TP53 mutations as a cell-intrinsic ibrutinib resistance factor in CLL. This novel finding may explain observed shorter remission durations for CLL patients with del17p/mTP53 undergoing ibrutinib therapy. Ex vivo data overall appear concordant with findings in patients treated with ibrutinib thus externally validating our approach and may allow for further refinements in ibrutinib therapy and response prediction.
Balasubramanian:Pharmacyclics LLC, an AbbVie Company: Equity Ownership; Janssen: Employment, Equity Ownership. Malek:Janssen Pharmaceuticals: Research Funding; Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership.
Introduction: The landscape of gene mutations in CLL prior to therapy is well-characterized. Comparatively less is known about gene mutations and their frequency in CLL patients that have relapsed ...after potent chemo-immunotherapy. Further, despite knowledge of subclonal TP53 mutations that enrich and likely drive CLL relapse in a fraction of cases, a comprehensive profile of gene mutations and their variant allele frequencies (VAFs) and clonal dynamics before and after chemo-immunotherapy in CLL is lacking.
Methods: We have procured paired pre-treatment and post-treatment samples from 53 CLL cases that had relapsed after chemo-immunotherapy and purified CLL CD19+ cells and CD3+ T-cells to purity with FACS. DNA from relapsed CLL was subjected to exome capture and whole exome sequencing (WES) at a mean coverage of 72-fold (range 52-102) and sequence data analyzed using three variant callers: MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Somatically acquired gene mutations occurring in 2 or more rCLL cases were confirmed by Sanger sequencing in relapsed CLL samples and also re-sequenced in pre-treatment samples. Genes with mutation frequencies ≥5% in rCLL underwent custom gene panel-based deep coverage re-sequencing in paired pre-treatment and post-treatment samples. Analysis of deep re-sequencing data was done using the Broad GATK HaplotypeCaller v3.3.0 in parallel with VarScan2. Selected low-level variants were measured using droplet digital PCR (ddPCR) that was adapted to detection of VAFs as low as 1/10,000.
Results: In CLL relapsed from potent chemo-immunotherapy, we detected mutated TP53, NOTCH1, SF3B1, XPO1, BIRC3, MYD88, NXF1, POT1, CACNA1E, CHD2, EGR2, FAM50A, FAT3, FBXW7, MGA, SAMHD1 and ZMYM3 with frequencies ≥5%. An additional 64 genes were mutated in 2/53 rCLL cases each. We performed ultra-deep panel-based re-sequencing of the 17 genes with frequencies ≥5% in 53 paired diagnosis and relapse samples, complementing selected variants with ddPCR validation to determine VAFs.
TP53 mutations constituted the most frequently enriched gene at relapse (7/53=13%) and the VAFs of all TP53 mutations substantially increased at relapse often from very minor subclones at diagnosis. Importantly, none of the clonal TP53 mutations in rCLL appeared directly induced by chemotherapy, but instead all were selected from pre-existing subclones. Similarly, subclonal mutations in SAMHD1 substantially enriched in four cases at relapse (4/53=8%) suggesting a role in resistance to chemotherapy.
The majority of NOTCH1 mutations (8/13) were already fully clonal at diagnosis without further enrichment at relapse. Three (3/13) subclonal NOTCH1 mutations substantially enriched at relapse, while two (2/13) clonal NOTCH1 mutations substantially decreased. The VAFs for SF3B1 mutations similarly demonstrated three patterns: i) clonal that remained clonal (4/10), ii) clonal that substantial declined and became subclonal at relapse (4/10), and, iii) subclonal that enriched but remained subclonal (2/10) at relapse. Of the 13 remaining genes, most demonstrated no consistent enrichment or depletion or remained subclonal at relapse.
Of biological interest, the genes FBXW7, MYD88, NOTCH1, NXF1, ZMYM3, XPO1, SF3B1 and POT1, were often already fully clonal in the pre-treatment samples, suggesting an early role in CLL pathogenesis rather than a later role in the development of CLL relapse.
Conclusion: In this large WES study focused on gene mutations in relapsed CLL paired with analysis of subclone dynamics using deep panel re-sequencing and ddPCR, we identify the genes TP53 and likely SAMHD1 as drivers of CLL relapse in 20% of cases. Multiple other genes previously implicated as CLL drivers did not consistently enrich at relapse. Further, a subset of the mutated genes was often already fully clonal pre-treatment; these genes likely serve an important role early in CLL pathogenesis that is independent of therapy. The majority of relapsed CLL in this cohort were not associated with the recurrent clonal emergence of known CLL driver mutations and based on the gene mutations frequencies reported here, much larger rCLL cohorts would need analysis to confirm possible additional low frequency gene drivers of rCLL.
Malek:Gilead Sciences: Equity Ownership; Abbvie: Equity Ownership; Janssen Pharmaceuticals: Research Funding.
Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active ...investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving.
Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL.
Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB.
To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC.
We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells.
We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis.
We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples.
Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL.
Malek:Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.
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