ALBERT

Description: *Contributed equally as first authors. **Contributed equally as senior authors. Recurrent mutations within EGR2, a versatile transcription factor involved in differentiation of hematopoietic cells, were recently reported in 8% of advanced-stage chronic lymphocytic leukemia (CLL) patients, where they appear to be associated with a worse outcome. EGR2 is activated through ERK phosphorylation upon B-cell receptor (BcR) stimulation, and we have previously shown that EGR2 -mutated CLL patients display altered expression of EGR2 down-stream target genes compared to wildtype (wt) patients, thereby pointing to a pathogenic role for EGR2 mutations in dysregulating BcR signaling. To gain further insight into the incidence and prognostic impact of EGR2 mutations in CLL, we screened samples from a well-characterized series of 1430 patients, either by Sanger sequencing (n=1019) or targeted deep-sequencing (n=370), both covering the recently reported EGR2 hotspot in exon 2. In addition, whole-exome data was available for an additional 43 patients. Different cohorts were included in our analysis ranging from 'general practice' CLL (33% IGHV-unmutated (U-CLL), 6% TP53 -aberrant (TP53abn), n=693), to adverse-prognostic CLL (89% U-CLL, 26% TP53abn, n=325), patients belonging to clinically aggressive stereotyped subsets #1-3 & #5-8 (n=342), patients relapsing after FCR therapy (n=41) and Richter transformed cases (n=31), thus reflecting the heterogeneous nature of CLL. Nineteen EGR2 mutations were detected by Sanger sequencing, while 22 additional mutations were identified with deep-sequencing using a 5% variant allele frequency (VAF) cutoff (median 39%, range 5.6-63.9%, median coverage 43,000X). With the exception of one in-frame deletion, all mutations were missense alterations located within the three zinc-finger domains. Significant enrichment of EGR2 mutations was observed in adverse-prognostic (18/325, 5.5%) and FCR-relapsing (4/41, 9.8%) CLL compared to the 'general practice' cohort (18/693, 2.6%, Figure 1A). A surprisingly low frequency was observed among clinically aggressive stereotyped subsets (5/342, 1.5%), although the cause for this observation is currently unknown. Finally, 2/31 (6.5%) cases with Richter transformation carried an EGR2 mutation. Of the 4 FCR-relapsing, EGR2 -mutated cases with available overtime samples, all demonstrated a significant expansion of the EGR2 -mutated clone at relapse (VAF-increase between 15-41%). In addition, subclonal levels of EGR2 hotspot mutations (VAF 0.5-5%) were detected in an additional 13/370 (3.5%) cases by deep-sequencing. The majority of EGR2 -mutated CLL patients (32/39, 82%) concerned U-CLL and the following aberrations co-occurred: 11q-deletions (n=10), TP53abn (n=6), NOTCH1 (n=3)or SF3B1 (n=3) mutations. EGR2 -mutated patients displayed a significantly worse overall survival compared to wt patients (median survival 59 vs. 141 months, p=0.003, using a conservative 10% VAF cutoff), and a poor outcome similar to cases with TP53abn (Figure 1B). In multivariate analysis (n=583), EGR2 status remained an independent factor (p=0.038), along with stage (p=0.048) and IGHV status (p

Description: Introduction: Phosphatidylinositol-3-kinase δ (PI3Kδ)-signaling provides key maintenance, proliferation, and survival cues during both normal and malignant B-lymphocyte development. Consequently, isoform-selective PI3Kδ inhibitors (PI3Kδi) have generated huge interest as a potential treatment for lymphoid malignancies. In particular, PI3Kδi demonstrate impressive clinical efficacy in combination with anti-CD20 monoclonal antibodies (mAbs) for relapsed chronic lymphocytic leukemia (CLL). However, these combinations function primarily to delay disease progression, but are not curative. With the ever-rising number of new targeted therapeutics, the challenge is to identify combinations that will ultimately deliver curative regimes. In order to guide these selections, a detailed mechanistic understanding is required. To date, only limited data are available regarding the exact in vivo therapeutic mechanism of PI3Kδi. Currently, studies identify immunomodulation, inhibition of BCR-, chemokine/cytokine-signaling, and induction of apoptosis as putative therapeutic mechanisms. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced cytotoxicity and determine the relative contribution toward in vivo therapeutic responses utilizing the Eµ-TCL1-Tg mouse model of CLL alongside human CLL samples. Methods: The molecular mechanisms of PI3Kδi alone or in combination with anti-CD20 mAbs were assessed using the Eµ-TCL1-Tg mouse model, an in vivo model system of CLL. To inhibit PI3Kδ, the δ isoform-selective inhibitor GS-9820 was chosen, as it is highly structurally related to idelalisib, and critically demonstrates improved pharmacokinetic properties in the mouse in comparison to idelalisib. In vitro GS-9820 IC50 are as follows: PI3Kδ 27 nM; PI3Kα 83,424 nM; PI3Kβ 14,899 nM; and PI3Kγ 15,606 nM. Assays to measure its effects on BCR-mediated kinase activation, chemokine signaling/chemotaxis, and cytokine- and cell-mediated support were performed. GS-9820 was administered in vivo at 10 mg/kg per os BID (formulated in 0.5% methylcellulose, 0.05% tween-80), once leukemias were detected, and maintained throughout the treatment period (GS-9820 Cmax 3114 nM, Ctrough 48.6 nM). Results: GS-9820 induced substantive in vitro cell death and disruptedBCR-mediated kinase activation, chemokine signaling/chemotaxis, and inhibited both cytokine- and cell-mediated support in murine (Eµ-TCL1) and human CLL cells. In vivo administration of GS-9820 imparted significant therapeutic responses in Eµ-TCL1-bearing animals, reducing leukemic burden by 75% and splenic tumor deposits by 66% 4 weeks posttreatment. GS-9820 appeared well tolerated in recipient animals with no obvious toxicity apparent (e.g. weight loss or behavioral symptoms). When in combination with anti-CD20 mAbs, GS-9820 extended leukemia depletion by several weeks. GS-9820 enhanced overall survival by 66% in comparison with vehicle control-recipient animals and enhanced the survival benefit of anti-CD20 mAb therapy. These therapeutic responses were associated with a 2-fold increase in expression of the pro-apoptotic BH3-only Bcl-2 family member Bim and a 3-fold increase in the extent of Bim/Bcl-2 interaction. Accordingly, Bim-/- Eµ-TCL1-Tg leukaemias exhibited profound resistance to PI3Kδi-induced cytotoxicity, were refractory to PI3Kδi in vivo, and failed to display combination efficacy with anti-CD20 mAbs. These findings informed the rational design of a GS-9820 + ABT199 (Venetoclax) complementary drug combination strategy. Combinations of GS-9820 and ABT199 were well tolerated with an absence of weight loss or altered behavioral symptoms. The GS-9820 + ABT199 combination effectively halted leukemia progression in vivo with increased efficacy compared to monotherapy regimes, resulting in a 90% reduction in leukemic burden at the end of the treatment period. Conclusions: Bim-dependent apoptosis represents the key in vivo effector mechanism for PI3Kδi in the Eµ-TCL1-Tg mouse model, both alone and in combination with anti-CD20 mAbs. As such, combinations of PI3Kδ and Bcl-2 inhibitors may represent an efficacious drug combination strategy. Disclosures Tannheimer: Gilead Sciences: Employment. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Cragg:Baxalta: Consultancy; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding; Gilead Sciences: Research Funding; GSK: Research Funding.

Description: The tumor microenvironment is characterized by multiple interactions of transformed malignant cells with non-transformed stroma or immune cells. Particularly macrophages play a pivotal role in this network determining disease progression and therapeutic response. In previous work we could show that macrophages are an essential mediator of therapeutic response in the synergistic response to the administration of the chemoimmunotherapy. The combination treatment strongly increases tumor clearance by repolarization of tumor-associated macrophages from a suppressive to an activated phenotypic state. Here, se analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. We attempted to disrupt DNA damage response pathway in lymphoma cells generated from the hMB humanized Double-Hit-Lymphoma model by knock-down of key elements like ATM, DNA-PK or p53. We could prevent the formation of the stimulatory cytokine release effect on macrophage phagocytic capacity. Here, p53 status displays a key regulatory role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wild-type cells, while not displaying enhancement in p53-deficient lymphoma cells. Addressing the treatment in vivo using the hMB model for modeling of Double-Hit Lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo. Using primary human CLL patient cells comparing both wild-type and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. Using a CLL mouse model by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice we could show that low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice. A similar effect was seen for primary multiple myeloma cells in response to daratumumab displaying significantly less ADCP of p53-deficient multiple myeloma cells. As for the mechanism of p53-defined interaction within the tumor microenvironment we subjected p53-wild-type and p53-deficient lymphoma cells for proteomic analysis. Here we could identify a significantly deregulated protein expression profile for exosome release in p53 deficient lymphoma cells. Verifying this finding by assessing size and frequency exosomes released by respective cell populations we reveal profound changes induced by p53 loss. Furthermore we could identify up-regulation of PD-L1 in p53-deficient cells. Blocking this checkpoint in the ADCP assay could significantly restore phagocytic capacity of macrophages and overall therapeutic response. In this work, we indicate that p53 functional status determines phagocytic function and therapeutic response to monoclonal antibodies. We can verify this finding in independent models in vitro and in vivo as in primary CLL and myeloma patient cells. We furthermore identify altered exosome profiles and checkpoint inhibitor expression in lymphoma cells as underlying mechanism of macrophage modulation. Finally our ongoing research offers possibility to reveal and tailor new combinatorial treatment approaches for chemo-refractory patients. Disclosures Wendtner: Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Hallek:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Pallasch:Gilead: Research Funding.

Description: Key Points Whole-exome sequencing of CLL patients who relapsed after FCR treatment revealed frequent mutations in RPS15. RPS15 mutations are likely to be early clonal events and confer poor prognosis.

Description: SF3B1 is a critical component of the RNA splicing machinery that achieves successful transcription and provides the functional diversity of protein species through alternative splicing. Recent studies have identified SF3B1 mutations in several tumours. In chronic lymphocytic leukemia (CLL) these mutations are associated with altered splicing, reduced survival and resistance to treatment. We therefore hypothesised that inhibition of the spliceosome may be an effective therapeutic strategy in SF3B1 mutated individuals. Initially, we utilized Spliceostatin A (SSA), an agent known to inhibit SF3B1 and alter gene splicing in various cell types. We showed that in CLL cells (SF3B1 WT and mutated samples), SSA treatment resulted in a dose-dependent increase in the un-spliced mRNA of DNAJB1 and RIOK3, two genes known to be regulated by SF3B1. We then assessed the ability of SSA to kill CLL cells with an SF3B1 mutant (n=12) or wild-type (n=28) genotype over a 24 hour period. CLL cells, regardless of SF3B1 mutational status, underwent apoptosis in a dose- (Figure A) and time-dependent manner (data not shown), with a mean IC50 of 5.1 nM. Interestingly, SSA induced more apoptosis in IGHV unmutated-CLL (U-CLL) compared to mutated-CLL samples (M-CLL) (p=0.037) and was active in 17p-deleted CLL (n=7), an aggressive disease sub-type that exhibits pronounced resistance to standard treatments. Normal B and T cells were significantly more resistant to SSA treatment than CLL samples purified from age-matched normal donors (B-cells p=0.006, T-cells p=0.001) and CLL derived T cells (p=0.003). SSA-induced apoptosis proceeded via a caspase-dependent mechanism (inhibited by ZVAD.FMK) coincident with elevations in Noxa. No changes in Bcl-2 or Bcl-xL were observed. Given the known ability of Noxa to interact with, and destabilise, Mcl-1, we next assessed the ability of SSA to modulate Mcl-1, and observed a substantial decrease in protein expression (Figure B, p=0.028). SSA treatment also altered the splicing of MCL-1; reducing the proportion of full length MCL-1L and increasing the amount of MCL-1s transcripts produced. The key role of Mcl-1 in regulating SSA-induced apoptosis was also observed in Ramos cells where over-expression of Mcl-1L, significantly protected them from SSA-induced apoptosis (p=0.001). Furthermore, in CLL cells exposed to CD40L and IL-4, factors known to induce Mcl-1 expression, SSA-induced apoptosis was significantly reduced (p=0.008). Taken together, these data implicate aberrant Mcl-1 splicing and Noxa upregulation in the apoptosis induced after SSA exposure. CD40L and IL-4 are proposed to be key factors preventing the apoptosis of CLL cells in the proliferative niche. Therefore, to explore possible strategies for overcoming this resistance, we investigated combining SSA with the Bcl-2/Bcl-xL inhibitor ABT-263 and showed that dual exposure to both inhibitors significantly increased apoptosis overcoming the protective effects of IL-4/CD40L (p=0.004; Figure C). In conclusion, our data shows that SSA induces apoptosis at nanomolar concentrations in CLL cells, independently of SF3B1 mutational status, through effects on Mcl-1. In combination with ABT-263, we show that SSA is particular active and can overcome the protective effects of CD40L and IL-4, suggesting promising clinical utility. Figure Legend. (A) Annexin V /PI analysis of CLL cells treated with SSA, (B) CLL cells treated with SSA for 24 h followed by immunoblotting for various proteins. (C) Drug combination studies with ABT-263 and SSA at a 1:1 ratio at 10 nM or 20 nM following IL-4/CD40L treatment. Figure Legend. (A) Annexin V /PI analysis of CLL cells treated with SSA, (B) CLL cells treated with SSA for 24 h followed by immunoblotting for various proteins. (C) Drug combination studies with ABT-263 and SSA at a 1:1 ratio at 10 nM or 20 nM following IL-4/CD40L treatment. Disclosures No relevant conflicts of interest to declare.

Description: Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and have been found disrupted at the genomic level in a spectrum of human tumors including hematological malignancies. In CLL, recurring genomic lesions targeting chromatin modifiers are emerging in the literature (Puente 2015, Nature) but their biological and clinical significance remain uncertain. We studied 802 CLL patients, divided into a discovery [n=261, sampled pre-treatment] and two validation cohorts [n=541, 431 pre-treatment and 110 ultra-high risk], using high-resolution SNP-arrays [n=572, Affymetrix SNP6.0 and HumanOmniS-8] and high-throughput re-sequencing [n=320, Haloplex and TruSeq] to identify genomic lesions targeting HMT genes. In our discovery cohort, we identified nine novel regions of copy-number changes; the prime finding was a recurrent deletion of chromosome 3p in 4% of patients between genomic positions 47.12-47.36Mb. This region included SETD2, KIF9 and KLHL18 (Fig 1A), with SETD2 being the most significantly under-expressed gene (p=0.001) in 3p-deleted [n=6] versus non-deleted cases [n=8]. Further validation in two independent cohorts showed that SETD2 deletions were enriched in ultra high-risk CLL and associated with loss of TP53 (p=0.003), genomic complexity (in TP53 wild-type cases, p=0.01) and chromothripsis. Next, we screened for somatic mutations in SETD2 using targeted re-sequencing and identified non-synonymous mutations in four (4.5%) discovery cases (p.D99G, p.W1306X, p.Q1545K, p.E1955Q), and 7/231 (3%) in the pre-treatment validation cases (p.A50T, p.P167L, p.E670K, p.M1742L, p.M1889T (x2), p.I2295M) (Fig 1B). Mutations were somatic in all samples tested [n=5]. To study the clonal nature of the SETD2 deletions, we assigned each genomic CNA with a relative copy-number by normalizing CNA intensity values from array features, and could infer that the 3p deletion was in the dominant clonal population in 14/21 cases with data available for analysis. Employing either the ABSOLUTE algorithm for our discovery cohort or manually correcting for tumor sample purity and local copy-number changes in our validation cohort, we observed that 10/11 SETD2 mutations exhibited a clonal cell fraction. These data strongly imply that SETD2 aberrations represent early clonal events in the pathobiology of CLL. Next, we extended our gene expression analysis to include additional wild-type, deleted, and mutated patients, showing reduced expression both in deleted and mutant cases (p=0.035), thus confirming haplo-insufficiency also for the SETD2-mutant cases. W e reviewed the DNA methylation status of the SETD2 gene body and promoter regions (15 & 9 CpG probes, respectively) from published data, and found no correlation between SETD2 methylation and RNA expression, suggesting that DNA methylation does not play a substantial role in regulating SETD2 expression in CLL. Finally, we analyzed the impact of SETD2 lesions on treatment-free survival (TFS) and overall survival (OS). For TFS and OS, we observed a significantly worse outcome (TFS: 44 vs. 105 months; p=0.004, OS: 85 vs. 199 months; p=0.002) in SETD2 deleted cases that were wild-type for TP53/ATM, compared to cases wild type for TP53/ATM/SETD2 (Fig 1D). Mutant SETD2 cases (wild-type for TP53/ATM) and those wild-type for TP53/ATM/SETD2 exhibited median TFS of 74 and 106 months respectively, differences that did not reach significance (p=0.1). Whilst these data suggest that SETD2 lesions may be clinically relevant, further investigations in larger materials are warranted to understand their full impact on survival. In conclusion, we report for the first time somatic deletions and mutations in SETD2, a gene found disrupted invarious human solid and hematological tumors, in ~7% of CLL patients requiring treatment. These are likely to be early clonal events and associate with TP53 dysfunction, genomic complexity and chromothripsis, with deletions enriched in ultra high-risk CLL. Figure 1. SETD2 lesions. A. SNP6.0 data for the del(3p) cases. B. Schematic diagram highlighting the prevalence and positioning of SETD2 mutations. C. Clonal cell fraction data for SETD2 and other gene mutations. D. Treatment-free survival (TFS). Figure 1. SETD2 lesions. A. SNP6.0 data for the del(3p) cases. B. Schematic diagram highlighting the prevalence and positioning of SETD2 mutations. C. Clonal cell fraction data for SETD2 and other gene mutations. D. Treatment-free survival (TFS). Disclosures Tausch: Gilead: Other: Travel support. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015. Hillmen:Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Dyer:Gilead: Research Funding; Roche Pharmaceuticals: Speakers Bureau; ONO Pharmaceuticals: Research Funding. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

Description: Richter's transformation (RT) is an aggressive lymphoma which occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL-phase involving TP53, CDKN2A, MYC, and NOTCH1, however a significant proportion of RT cases lack CLL-phase associated events. Here, we report that high levels of AKT phosphorylation occurs both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in RT patients. Genetic over-activation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and DLBCL. Multi-omics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed a S100-protein-defined subcluster of highly aggressive lymphoma cells, which developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation towards aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Description: * These authors contributed equally Introduction Causes of death and, in particular, deaths due to infection have not been widely studied in randomised trials in chronic lymphocytic leukaemia (CLL). With long-term follow-up (median 13 years), we were able to examine the cause of death in 600/777 patients in the LRF CLL4 trial. Blood samples taken at randomization from 499 patients were available, allowing us to examine the relationship between deaths due to infection and a large panel of genes which are commonly mutated in CLL. Several gene mutations have been linked to earlier death in the LRF CLL4 trial, including mutations of TP53, NOTCH1, SF3B1, EGR2 and MAPK-ERK (Gonzalez et al, J Clin Oncol 2011; 29:2223-9; Oscier et al, Blood 2013; 121:468-75; Young et al, Leukemia 2017; 31:1547-54; Blakemore et al, Leukemia 2020; 34:1760-4). In this study we aimed to identify gene mutations which were specifically associated with death due to infection. Methods In LRF CLL4 patients were randomized between 1999-2004 to receive chlorambucil or fludarabine, with or without cyclophosphamide. Follow-up continued until September 2016. Causes of death were assessed centrally by the principal investigator. Results In the LRF CLL4 trial 614 of 777 patients (79%) died before the end of follow-up. The cause of death was known in 600 patients. Deaths tended to be multifactorial, but infection was a cause of death in 258 patients (43%). Fatal infections were pneumonia (67%), and/or sepsis (38%) and/or opportunistic infections such as aspergillus (11%). Patients who died of infection were more likely than those who died of other causes to have received more than one line of treatment and to have died in the winter months (Table 1). Mutations of BRAF, FBXW7, NRAS and XPO1 were significantly associated with death due to infection versus other deaths. However, with multiple hypothesis testing, NRAS was the only genetic mutation to survive a false discovery rate (FDR) q-value = 0.05 (odds ratio: 17, P = 0.0004). No other significant differences were found between patients who died of infection versus those whose death did not have an infectious cause. In particular, the rate of deaths due to infection was not influenced by other demographic or laboratory factors, nor by the randomised treatment, the response to treatment, or the size/experience of the CLL treatment centre. In multivariate analysis the factors most significantly associated with death from infection versus all other deaths were mutations of the BRAF, FBXW7, NRAS and XPO1 genes (Table 1). Of the 499 patients in the trial for whom gene mutation data were available, 73 (15%) carried one or more of the four gene mutations BRAF (6%), FBXW7 (2%), NRAS (2%) and XPO1 (6%) (Table 2). Only six of these 73 remained alive. Death was caused by infection in 46/67 assessable patients (69%) who had a mutation of one or more of these four genes versus only 129/333 patients (39%) without any of these mutations (odds ratio: 3.46 [95% C.I. 1.98-6.07] P