Abstract: Background: The MYC proto-oncogene encodes a DNA-binding factor that can induce widespread changes in gene expression profiles (GEP). Activation of MYC is a hallmark of aggressive lymphomas and frequently observed in Richter transformation of CLL. In contrast, the role of MYC-related pathogenic networks is less clearly defined in untransformed CLL. Aims: We hypothesized that MYC activation in CLL could lead to specific GEP associated with aggressive disease. We combined the analysis of genomic copy number alterations (CNA) and GEP involved in MYC pathway activation on specimens from patients registered on the CLL8 trial (front line therapy FC vs. FCR). Methods: GEP were derived from CD19-enriched CLL samples (n=337, Human Exon 1.0 ST, Affymetrix) and analysis of CNA was performed based on availability of DNA (n=309, Human SNP Arrays 6.0, Affymetrix). Sample work-up upon trial registration included FISH and TP53 mutation analysis. Results: Genomic gains involving the MYC locus on 8q24.21 were observed in 4.5% of cases. To test the hypothesis of specific GEP associated with MYC activation, we explored the distribution of cases with MYC gain using an unsupervised approach on GEP. After consensus clustering (k=6 clusters) of variably expressed genes (SD 〉 0.5), cases with MYC gain were non-randomly distributed and showed a characteristic pattern. Preferential enrichment was observed in one cluster ("MYC-CNA" group, comprising 40% of all cases) with 64% of MYC gains. Gene set enrichment analysis (GSEA) confirmed overrepresentation of MYC target genes (gene set: HALLMARK_MYC_TARGETS_V1, FDR 〈 0.05) in MYC-CNA and a second cluster, denoted as MYC endogenous activation cluster ("MYC-EA" group, 16% of cases). Conversely, a large cluster, which was most distant to MYC-CNA, did not show significant enrichment in GSEA for MYC target genes or for CNA and was defined as "MYC-silent" reference cluster (comprising 30% of cases). Other potential elements contributing to the regulation of MYC networks, included enrichment of TP53 alterations in both MYC clusters compared to the MYC-silent cluster (17.5% vs. 6%, p=0.015, Fisher`s exact test). We also observed frequent gains of chromosome 2p, involving NMYC on 2p24.3, in both MYC clusters. Losses of the MYC repressors MNT on 17p13.3, MGA on 15q15.1 and PRDM1 on 6q21 also constituted frequent events in the MYC-CNA cluster. Overall, CNA affecting MYC, NMYC and the MYC repressors were more frequent in MYC-CNA (41 in 127 cases) compared to MYC-silent cluster (15 in 93 cases) (p=0.03, Mann Whitney). In addition, expression of the MYC repressor BCL6 was downregulated in MYC-CNA compared to the MYC-silent cluster (fold change 1.5, q 〈 1e-07). MYC protein overexpression was observed by Western blot densitometry in cases without the described CNA, both in MYC-CNA (n=11 cases tested) and MYC-EA (n=7 cases tested), confirming independent activation. Activation of PI3K-AKT and RAS-ERK-signaling was a prominent feature in both MYC clusters. Strong discrepancy between both MYC clusters was observed for cell cycle regulation with changes implicating either increased proliferation in MYC-CNA or cell cycle arrest in MYC-EA. PFS was different when comparing both treatment arms in MYC-CNA (HR 0.55 (95%CI 0.37-0.82), p=0.003) and MYC-EA (HR 0.30 (95%CI 0.15-0.60), p 〈 0.001) with PFS rates at 5 years of 15% (FC) vs. 38.6% (FCR) for MYC-CNA and 17.9% (FC) vs. 57.6% (FCR) for MYC-EA. In contrast, the MYC-silent cluster showed a better outcome compared to the MYC clusters when treated with FC and no benefit from the addition of rituximab with PFS rates at 5 years of 43.1% (FC) vs. 42.9% (FCR) (p=0.56). Median OS was significantly different for treatment arms in MYC-EA and, compared to all other clusters, showed shortest median OS for FC treatment with OS rates at 5 years of 47.9% and strongest benefit for the addition of rituximab with 80.5% (FCR) (HR 0.32 (95%CI 0.13-0.79), p=0.009). Conclusion: MYC pathway alterations were frequently observed in treatment-naive CLL and may involve various mechanism such as CNA affecting MYC and its repressors, TP53 defect or sole transcriptional changes. Cases with MYC activation may be segregated based on cell cycle checkpoint deregulation and consecutive proliferative capacity. Clusters with MYC activation had an inferior clinical course when treated with FC but, when adding rituximab, both MYC-CNA and MYC-EA showed a significant improvement for outcome. Disclosures Bahlo: Roche: Honoraria, Other: Travel Grants. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wenger:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership, Other: Ownership interests PLC. Tausch:AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Bullinger:Bayer Oncology: Research Funding; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Fischer:Roche: Other: Travel support. Hallek:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Boehringer Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stilgenbauer:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: 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; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: Apoptosis is controlled by the expression levels and interplay of pro- and anti-apoptotic BCL-2 family proteins. The specific BCL-2 inhibitor Venetoclax (VEN) showed high efficiency in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Despite its high efficiency in CLL and MCL, refractory disease can develop. BCL-2 mutations have been described to mediate resistance in CLL cases, however these mutations are only found in a proportion of VEN resistant cases and in a fraction of cells. In order to design alternative therapeutic strategies to overcome drug resistance, a better understanding of the mechanisms mediating resistance to VEN is necessary. VEN-resistant (VEN-R) MCL cell lines (MINO and MAVER-1) were generated by chronic exposure to increasing amounts of VEN (up to 3µM). A significant and stable upregulation of BCL-XL mRNA and protein was seen in the MINO and MAVER-1 resistant cell lines (2 and 4 fold increase in mRNA and 2.6 and 4.5 fold increase in protein, respectively). We used BH3 profiling in combination with VEN treatment for 4h to investigate the differences in anti- and pro-apoptotic signaling in parental and VEN-R cell lines. Additionally, sensitivity to VEN was restored upon shRNA-mediated knockdown of BCL-XL. These results confirmed the importance of BCL-XL upregulation in mediating resistance. Furthermore, we did not detect mutations in BCL-2 upon resistance to VEN via targeted NGS, which is in contrast to results obtained in VEN-R CLL patients (Blombery et al., Cancer Discovery 2019 and Tausch et al., Hematologica 2019). However, the results obtained by dynamic BH3-profiling (VEN treatment in combination with BH3 Profiling) suggest that increase in BCL-XL is most likely not the only alteration necessary to render cells resistant to VEN. In addition, reduced activation of pro-apoptotic proteins like BAX and BAK might contribute to resistance to VEN. In order, to investigate if VEN resistance can be overcome by drug mediated inhibition of BCL-XL we used different therapeutic approaches. Combinational treatment with the BCL-XL inhibitor A-1331852 and VEN or the single treatment with Navitoclax, a combined inhibitor of BCL-2, BCL-W and BCL-XL for 48h reduced cell viability in VEN-R MINO and MAVER-1 cell lines. Furthermore, BDA-366, a BH4 domain BCL-2 inhibitor effectively reduced the cell viability after 48h of treatment in a dose dependent manner in both parental and VEN-R cell lines. The binding of BDA-366 to the anti-apoptotic BCL-2 protein leads to a conformational change into a pro-apoptotic molecule by the exposure of the BH3 domain of the protein. Despite mediating apoptosis in a TP53-independent manner, VEN treatment in CLL has been associated with inferior outcome in the presence of TP53 aberrations. In order to address the role of TP53 dysfunction in mediating resistance to VEN, we generated p53 knock out cell lines (N=2) by CRISPR/Cas9 gene editing. This significantly decreased the sensitivity to VEN compared to p53 WT cell lines. Additionally, the sensitivity to BDA-366 was significantly reduced upon knockout of p53, suggesting an interference of p53 downstream of BCL-2. Overall, VEN resistance is mediated by a permanent increase in BCL-XL mRNA and protein level in MCL. Importantly, BDA-366, which converts the anti-apoptotic BCL-2 molecule into a BAX-like death molecule, could be a potential alternative treatment strategy for BCL-2 dependent cancers even when resistant to VEN. Despite mediating apoptosis in a p53 independent manner, VEN seems to be less effective in p53 deficient cells, underlining the importance of further investigations of treatment combinations in these groups. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau. Schneider:Celgene: Other: travel grant.

Abstract: Chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab (FCR) can induce long-term remissions in patients with chronic lymphocytic leukemia. Treatment efficacy with Bruton's tyrosine kinase inhibitors was found similar to FCR in untreated chronic lymphocytic leukemia patients with a mutated immunoglobulin heavy chain variable (IGHV) gene. In order to identify patients who specifically benefit from FCR, we developed integrative models including established prognostic parameters and gene expression profiling (GEP). GEP was conducted on n=337 CLL8 trial samples, “core” probe sets were summarized on gene levels and RMA normalized. Prognostic models were built using penalized Cox proportional hazards models with the smoothly clipped absolute deviation penalty. We identified a prognostic signature of less than a dozen genes, which substituted for established prognostic factors, including TP53 and IGHV gene mutation status. Independent prognostic impact was confirmed for treatment, β2-microglobulin and del(17p) regarding overall survival and for treatment, del(11q), del(17p) and SF3B1 mutation for progression-free survival. The combination of independent prognostic and GEP variables performed equal to models including only established non-GEP variables. GEP variables showed higher prognostic accuracy for patients with long progression-free survival compared to categorical variables like the IGHV gene mutation status and reliably predicted overall survival in CLL8 and an independent cohort. GEP-based prognostic models can help to identify patients who specifically benefit from FCR treatment. The CLL8 trial is registered under EUDRACT-2004- 004938-14 and clinicaltrials gov. Identifier: NCT00281918.

Abstract: Targeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of IGF1R in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor–resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3β. In human CLL, high IGF1R expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor–resistant tumors in vitro and in vivo.

Abstract: In chronic lymphocytic leukemia (CLL) short telomeres are associated with other adverse prognostic factors and poor survival. We and others have described association of telomere length with genomic complexity and clonal evolution in CLL. To understand if telomere shortening rather than being only a marker of cell proliferation, could also functionally contribute to disease progression, we generated Terc knock out in the Eµ-TCL1 murine CLL model (Eµ-TCL1 mTerc-/- mice). Comparison of the Eµ-TCL1 mTerc-/- mice from the generations G1, G2 and G3 with that of Eµ-TCL1 (TCL1+) did not show a difference in disease initiation, progression as well as survival even though a significant decrease in telomere length of tumor cells was observed with increasing generations. Of interest, the Eµ-TCL1 mTerc-/- G3 tumors (n=8; G3) more frequently showed defective DNA damage response compared to TCL1+ tumors (n=8), as analysed by changes in phosphorylation of gamma-H2AX, ATM and p53 measured by FACS at 1, 3, 6, 16 and 24 hours after gamma irradiation. Despite this predisposition to undergo genomic complexity, the G3 mice showed no difference in disease development compared to TCL1+ mice. Therefore we investigated if cell extrinsic factors in Terc-/- mice could affect tumor development. The Terc-/- microenvironment is known to be restrictive to B and T-lymphopoiesis and could hence inhibit CLL development. To study the impact of Terc-/- microenvironment, splenic tumors from the Eµ-TCL1 mTerc-/- G3 (n=11; G3) mice were transferred into syngeneic Terc+/+ C57Bl6 mice and compared with that of TCL1+ (n=11). No significant difference in disease burden and survival was observed between these 2 cohorts, indicating that there is no adverse influence of the Terc-/- microenvironment on the tumor growth in the Eµ-TCL1 mTerc-/- mice. Further, we hypothesized that the telomere length in the G3 mice may not be short enough to induce genomic instability and enhance disease aggressiveness. The mice were thus crossed to obtain the Eµ-TCL1 mTerc-/- generation G4 (G4). Though the survival of these mice were similar to that of Eµ-TCL1 controls (median survival :49 vs. 51 weeks; P=0.301), the G4 mice showed significantly decreased disease burden as measured by spleen weight, liver weights and tumor cell fraction. The G4 mice showed decreased fertility and hence further crosses to generate G5 were not performed. However, further shortening of telomeres was achieved by serially transplanting the tumors from G3 into syngeneic recipient mice for 2 generations. Telomere length of the tumor cells analysed by Q-PCR showed a significant decrease with increasing transfer rounds, compared to primary TCL1+ tumors. Interestingly, the 2nd round transfer of the G3 tumors led to a less severe disease and significantly longer survival of the recipient mice compared to 2nd round TCL1+ tumors transplants (median survival from date of transplantation: 15 vs. 7 weeks; P=0.030), indicating that cell intrinsic factors in the G3 tumors hamper proliferation of these cells. We finally analyzed if absence of telomerase could mask disease aggressiveness of the G3 tumors by crossing the G3 mice with wildtype Terc+/+ mice. The resulting TCL1+ mTerc+/- G4 mice showed an increased telomerase activity but had significantly shorter telomere length compared to TCL1+ mice. Strikingly, the TCL1+ mTerc+/- G4 mice showed faster disease development and significantly shorter survival (median survival: 42 vs. 51 weeks; p 〈 0.001) as compared to TCL1+ mice. The aggressiveness of the TCL1+ mTerc+/- G4 tumors was further verified using adoptive transfer into syngeneic mice, and mice transplanted with these tumors showed a significantly shorter survival as compared to TCL1+ or G3 tumors. In summary, the TCL1+ mTerc-/- mice crossed through generations G1 to G4 did not show a difference in disease initiation, progression or survival despite significant shortening of telomeres. However, the tumors from G3 had defective DDR, indicating a potential for accumulating genetic aberrations and clonal evolution. But the absence of functional telomerase decreased the growth potential of these genomic instable tumors. Reconstitution of telomerase in G3 mice resulted in aggressive tumors with short telomere length and could therefore be a valuable murine model for genomic instability and aggressive CLL. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Schneider:Celgene: Other: travel grant. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.