Simultaneous Inhibition of PI3Kd and PI3Ka Induces ABC-DLBCL Regression by Blocking BCR- Dependent and -Independent Activation of NF-kB and AKT

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1 Article Simultaneous Inhibition of PI3Kd and PI3Ka Induces ABC-DLBCL Regression by Blocking BCR- Dependent and -Independent Activation of F-kB and AKT Graphical Abstract Authors Juliane Paul, Maurice Soujon, Antje M. Wengner,..., Soon Thye Lim, Karl Ziegelbauer, ingshu Liu Correspondence In Brief Paul et al. reveal that ABC-DLBCL expresses high levels of PI3Ka and PI3Kd, leading to downstream activation of both F-kB and AKT signaling. Treatment with the dual PI3Ka/d inhibitor copanlisib has strong anti-tumor activity and synergizes with the BTK inhibitor ibrutinib, causing tumor remission in DLBCL models. Highlights d Dual PI3Ka/d inhibition is required to block AKT and F-kB activation in ABC-DLBCL d d d PI3K regulates BCR-dependent and -independent F-kB activation via p-ikba and ciaps PI3Ka/d inhibitor copanlisib induced tumor regression in MYD88 mut ABC-DLBCL in vivo Ibrutinib and copanlisib combination led to sustained complete response of DLBCL Paul et al., 2017, Cancer Cell 31, January 9, 2017 ª 2017 Elsevier Inc.

2 Cancer Cell Article Simultaneous Inhibition of PI3Kd and PI3Ka Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of F-kB andakt Juliane Paul, 1,5 Maurice Soujon, 1,5 Antje M. Wengner, 1 Sabine Zitzmann-Kolbe, 1 Andrea Sturz, 1 Katja Haike, 1 Koh Hui Keng Magdalene, 2 Sze Huey Tan, 3 Martin Lange, 1 Soo Yong Tan, 2,6 Dominik Mumberg, 1 Soon Thye Lim, 4 Karl Ziegelbauer, 1 and ingshu Liu 1, * 1 Bayer AG, Drug Discovery Oncology, Muellerstrasse 178, Berlin, Germany 2 Advanced Molecular Pathology Laboratory, Singapore Health Services Pte Ltd, 20 College Road, Singapore, Singapore 3 Clinical Trials and Epidemiological Sciences, ational Cancer Centre Singapore, 11 Hospital Drive, Singapore, Singapore 4 Office of Education, Duke-US Graduate Medical School, 8 College Road, Singapore, Singapore 5 Co-first author 6 Present address: Department of Pathology, ational University of Singapore, 5 Lower Kent Ridge Road, Singapore, Singapore *Correspondence: ningshu.liu@bayer.com SUMMARY Compared with follicular lymphoma, high PI3Ka expression was more prevalent in diffuse large B cell lymphoma (DLBCL), although both tumor types expressed substantial PI3Kd. Simultaneous inhibition of PI3Ka and PI3Kd dramatically enhanced the anti-tumor profile in ABC-DLBCL models compared with selective inhibition of PI3Kd, PI3Ka, or BTK. The anti-tumor activity was associated with suppression of p-akt and a mechanism of blocking nuclear factor-kb activation driven by CD79 mut, CARD11 mut, TFAIP3 mut,or MYD88 mut. Inhibition of PI3Ka/d resulted in tumor regression in an ibrutinib-resistant CD79B WT /MYD88 mut patient-derived ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79B mut -ABC-DLBCL to show a robust response to PI3K and BTK inhibitor monotherapies. A combination of ibrutinib with the PI3Ka/d inhibitor copanlisib produced a sustained complete response in vivo in CD79B mut /MYD88 mut ABC-DLBCL models. ITRODUCTIO Diffuse large B cell lymphoma (DLBCL) is the most common aggressive non-hodgkin lymphoma (ahl). Although the introduction of rituximab has improved the outcome of patients with DLBCL, disease progresses in approximately 30% 40% cases (Sehn et al., 2005). Patients with activated B cell-like (ABC)-DLBCL show less favorable clinical outcomes and are characterized by a constitutively activated nuclear factor-kb (F-kB) pathway, which promotes tumor proliferation and survival and confers chemotherapy resistance (Turturro, 2015). Chronic B cell receptor (BCR) signaling and mutations in CD79A/B, CARD11, TFAIP3, and MYD88 most commonly drive F-kB activation in DLBCL patients (Davis et al., 2010; Lenz et al., 2008; go et al., 2011). Constitutive activation of BCR in ABC-DLBCL leads to activation of the F-kB pathway via a cascade of kinases, such as spleen tyrosine kinase (SYK), Bruton s tyrosine kinase (BTK), protein kinase C-b (PKCb) (Buggy and Elias, 2012; Dal Porto et al., 2004; Kurosaki, 2011; Macias-Perez and Flinn, 2013). Inhibition of BTK with ibrutinib (PCI-32765), a potent irreversible inhibitor, has demonstrated an overall response rate (ORR) of Significance We found that high expression of PI3Ka in addition to PI3Kd in ABC-DLBCL modulates not only p-akt but also nuclear FkB activity, which is associated with resistance to PI3Kd selective inhibition. Simultaneous inhibition of PI3Ka/d by copanlisib showed promising activity in ibrutinib-resistant ABC-DLBCL with CD79A/B WT /MYD88 mut, TFAIP3 mut, or CARD11 mut. A Combination of PI3Ka/d and BTK inhibitors demonstrated potential for ibrutinib-responsive, insensitive, and/or relapsed ABC-DLBCL to achieve sustained complete response and prevent tumor recurrence by blocking rebound activation of BTK, AKT, and F-kB. Thus, our findings provide additional insights on intrinsic and acquired resistance mechanisms of selective PI3Kd and BTK inhibitors and the rationale for clinical testing of PI3K inhibitors with a specific isoform profile in combination for the treatment of ABC-DLBCL. 64 Cancer Cell 31, 64 78, January 9, 2017 ª 2017 Elsevier Inc.

3 A B C E F D G (legend on next page) Cancer Cell 31, 64 78, January 9,

4 41% in ABC-DLBCL. However, patients with CD79B WT and MYD88 mut or CARD11 mut did not respond to ibrutinib (Wilson et al., 2015), indicating the need for therapies targeting BCR-independent activation of F-kB induced by these mutations. Class I PI3Ks comprise four isoforms: PI3Ka, PI3Kb, PI3Kg, and PI3Kd. Activation of the PI3K signaling pathway has been demonstrated in numerous human malignancies, including indolent HL (ihl) and ahl (Dasgupta et al., 2005; Kloo et al., 2011). The recent approval of the PI3Kd-selective inhibitor idelalisib for the treatment of relapsed or refractory CLL in combination with rituximab, follicular lymphoma (FL), and small lymphocytic leukemia (SLL) confirmed the utility of PI3K inhibitors in these HL subtypes. However, in contrast to the significant clinical benefit in ihl (59% ORR), idelalisib did not show activity in DLBCL in a phase I study (Furman et al., 2010; Gopal et al., 2014; Kahl et al., 2014). The contrasting clinical outcomes of idelalisib in ihl and DLBCL question the roles of PI3K in these tumors. Furthermore, the inactivity of ibrutinib in a subset of DLBCL underscores the need for new therapies. In this study, we investigated the expression and functional significance of class I PI3K isoforms with the aim of identifying treatment strategies for ABC-DLBCL, a cancer type involving multiple oncogenic signaling pathways. RESULTS Expression of PI3Ka Is Significantly Increased in DLBCL Compared with FL Expression analysis of PI3K isoforms (p110a, p110b, p110d, and p110g) and PTE was conducted with primary tumors from 45 FL patients and 45 DLBCL patients by immunohistochemistry (IHC). PI3Kd high was found to be predominant in both FL (39/ 45, 87%) and DLBCL (43/45, 96%) (Figures 1A and 1B). In contrast, only 8/45 (18%) patients with FL displayed PI3Ka high and also PI3Kd high. PI3Ka expression level correlated with late stage of the disease (p = 0.06, chi-squared test) and with high Follicular Lymphoma International Prognostic Index (FLIPI) risk score (p = 0.01), but not with histologic grade (Table 1). Therefore, the expression of PI3Ka might play an important role in disease progression and portend a poor prognosis. otably, the incidence of PI3Ka high was greatly increased in DLBCL (62%) compared with FL (18%). The incidence of PI3Kb high and/or PI3Kg high was relative low in both FL (4% and 20%, respectively) and DLBCL (20% and 22%, respectively). PTE loss was found in 24% of FL and 27% of DLBCL patients. In addition, the expression profiles of PI3Ks and PTE were different between germinal center B cell-like (GCB)-DLBCL and ABC-DLBCL subtypes. ABC-DLBCL showed a lower incidence of PTE loss (14%) and PI3Ka high (57%) compared with GCB-DLBCL (70% and 80%, respectively). The representative expression features of ABC subtypes are depicted in Figure 1C. In summary, the finding of increased incidence of PI3Ka high in DLBCL sheds light on the molecular basis of the intrinsic resistance of DLBCL to PI3Kd inhibition observed in the clinic. Five ABC-DLBCL cell lines covering the representative oncogenic mutations (CD79 mut, CARD11 mut, TFAIP3 mut, and MYD88 mut )(Lohr et al., 2012) were subjected to expression analysis of PI3K isoforms and PTE (Figure 1D). The PI3Ka:PI3Kd expression ratio was high in HBL-1 and U2932, low in TMD-8 and OCI-Ly3, and medium in WSU-DLCL2. Furthermore, these five cell lines also covered ibrutinib-sensitive (TMD-8, HBL-1, and WSU-DLCL2) and resistant (OCI-Ly3 and U2932) features, and therefore were used for functional characterization of PI3K and BTK inhibition in ABC-DLBCL. Simultaneous Inhibition of PI3Ka/d Is Essential for Potent and Broad Anti-tumor Activity in ABC-DLBCL The PI3Ka-selective inhibitor BYL-719, the PI3Kd-selective inhibitor idelalisib, the PI3Ka/d predominant inhibitor copanlisib, and the BTK inhibitor ibrutinib were selected as clinically relevant reference agents for functional analysis in DLBCL cells (Figure S1A and Table S1) (Liu et al., 2013b; Scott et al., 2016). Anti-proliferative activity was determined using a cell viability assay. To dissect multiple signaling inputs (e.g., from different PI3K isoforms) to cell proliferation and survival, we used two parameters to address the activity of the inhibitors: (1) the half maximal inhibitory concentration (IC 50 ) to indicate inhibition of predominant proliferative signaling; and (2) IC 90 to indicate adequate tumor growth inhibition (Figure 1E). The three PI3K inhibitors revealed differential activity profiles: copanlisib demonstrated potent activity (IC 50 values ranging from 12 to 21 nm) in both ibrutinib-sensitive TMD-8 and HBL-1 (CD79B mut / MYD88 mut ) and ibrutinib-insensitive WSU-DLCL2 (WT CD79 and F-kB) or -resistant OCI-Ly3 (CARD11 mut ) and U2932 (TFAIP3 mut ) cell lines. Interestingly, idelalisib showed the most potent activity in the PI3Kd prominent TMD-8 tumor cells (IC 50 = 50 nm) where BYL-719 was 30-fold less active. In contrast, idelalisib was inactive in HBL-1 and U2932 (high PI3Ka:PI3Kd), while BYL-719 was >10-fold more active (IC 50 = 0.61 mm and 1.03 mm, respectively). In WSU-DLCL2 cells Figure 1. Expression and Functional Activity of PI3K Isoforms and PTE in HL (A C) The expression of PI3K isoforms and PTE was characterized by immunohistochemistry (IHC) analysis using antibodies selectively against PI3Ka, PI3Kb, PI3Kd, and PI3Kg. IHC was conducted using formalin-fixed paraffin-embedded samples from 45 FL (A) and 45 DLBCL (B) patients. The overall incidence (Aa and Ba) and individual relative expression (Ab and Bb) of each PI3K isoform and PTE in FL and DLBCL was determined using an intensity grading of 0, 1+, 2+ and 3+ for negative, low, moderate, and high expression, respectively. (C) IHC staining of PI3Ka, PI3Kd, and PTE in representative ABC-DLBCL samples. (D) Western blot analysis of PI3K isoforms, PTE and BTK, in five ABC-DLBCL cell lines and their corresponding genetic mutation status. (E) Cell viability was assessed using a CellTiter-Glo Luminescent Assay (Promega). The effect of ibrutinib, copanlisib, idelalisib, and BYL-719 was determined at 72 hr after compound treatment versus DMSO treatment (0% inhibition) and the value at 0 hr was used as 100% inhibition. The IC 50 (Ea) and IC 90 (Eb) of each compound in the tumor cell lines with a low, medium, and high ratio of PI3Ka/PI3Kd expression were generated from one representative experiment with triplicates on each data point. *Greater than indicated maximum concentration tested. (F) Inhibition of the PI3K pathway was assessed by p-akt (S473) and p-4e-bp1 (S65) and apoptosis induction by cleaved PARP using western blot in TMD-8 cells 24 hr after BYL-719 and/or idelalisib treatment. (G) Apoptosis induction was measured by cleaved PARP using western blot in TMD-8 cells at 24 hr after copanlisib or ibrutinib treatment. See also Figure S1, Tables S1 and S2. 66 Cancer Cell 31, 64 78, January 9, 2017

5 Table 1. Correlation of PI3Ka Expression and Disease Characteristics Frequency (%) Disease Characteristics Total ( = 45) PI3Ka Score 3+ (n = 8) PI3Ka Score 2+ (n = 19) PI3Ka Score 1+ or 0 (n = 18) p Value Stage 0.2 (0.06 a ) I and II 16 (35.6) 1 (12.5) 6 (31.6) 9 (50.0) III and IV 29 (64.4) 7 (87.5) 13 (68.4) 9 (50.0) Age group <60 34 (75.5) 6 (75.0) 28 (75.7) >60 11 (24.4) 2 (25.0) 9 (24.3) FLIPI group 0.03 b (0.01 a,b ) Low/intermediate (0 2) 30 (66.7) 3 (37.5) 11 (57.9) 16 (88.9) High (>3) 14 (31.1) 4 (50) 8 (42.1) 2 (11.1) ot evaluated 1 (2.2) 1 (12.5) 0 (0) 0 (0) Histologic grade 1 and 2 26 (57.8) 5 (62.5) 21 (56.8) 3A 18 (40.0) 3 (37.5) 15 (40.5) 3B 1 (2.2) 0 (0) 1 (2.7) Disease characteristics are summarized as frequency and percentage. Comparisons of disease stage and FLIPI group by PI3Ka scores (classified into three ordinal groups as 0/1+, 2+, and 3+) were performed using Fisher s exact test. The chi-squared test for trend was used to test if there was a linear trend relating disease stage and/or FLIPI risk group to PI3Ka score. A p value less than 0.05 was considered statistically significant. All analyses were performed in STATA version p value was calculated using Fisher s exact test. a p Value for test of trend was calculated using the chi-squared test for trend. b p Values were calculated excluding the patient not evaluable for FLIPI. (medium PI3Ka:PI3Kd), idelalisib and BYL-719 showed similar potency (2.27 mm versus 2.67 mm). otably, although idelalisib and copanlisib have equal cellular potency against PI3Kd (IC 50 = 7.4 nm versus 7.9 nm, Table S1; Lannutti et al., 2011), the anti-proliferative activity of idelalisib is in general much lower compared with copanlisib. Furthermore, in contrast with copanlisib, both idelalisib and BYL-719 failed to reach 90% growth inhibition except idelalisib in TMD-8 cells (IC 90 = 2.53 mm). To confirm that the inhibition of PI3Kd or PI3Ka alone is not sufficient to reach substantial growth inhibition, we examined simultaneous inhibition of PI3Ka and d. Indeed, the combination of idelalisib and BYL-719 reached greater inhibition of p-akt and downstream p-4e-bp1, and induced substantial cleaved PARP at lower concentrations compared with each single agent alone (Figure 1F). Isobologram analysis of idelalisib and BYL-719 in the cell viability assay indicated a strong synergistic combination effect, which led to a dramatic decrease in the concentrations required for reaching IC 90 compared with single-agent activity (Table S1). In contrast, selective PI3Kb inhibition by TGX-221 could not suppress the F-kB and PI3K pathways assessed by interkeukin-10 production (Figure S1B) and p-4e-bp1 (Figure S1C), respectively. Furthermore, adding TGX-221 to BYL- 719 and idelalisib did not further potentiate pathway inhibition (p-4e-bp1) and apoptosis induction (Figure S1C). The cellular inhibitory profile of copanlisib and ibrutinib was also distinct. Thus, despite comparable IC 50 values of copanlisib and ibrutinib in TMD-8 and HBL-1 cell lines, the IC 90 values of ibrutinib were much higher than that of copanlisib in both TMD-8 (871 nm versus 18 nm) and HBL-1 (5410 nm versus 137 nm) (Figure 1E). These data suggest that copanlisib adequately inhibited several key survival pathways, while the inhibition of BTK by ibrutinib might not be sufficient to reach complete tumor cell growth inhibition. This hypothesis was supported by substantial induction PARP cleavage by copanlisib at a concentration of 300 nm (below the clinical C max ; Patnaik et al., 2016) while ibrutinib up to 1 mm (above the clinical C max ; Advani et al., 2013) was incapable of inducing apoptosis (Figure 1G). Collectively, dual inhibition of PI3Ka and PI3Kd by copanlisib or a combination of idelalisib and BYL-719 led to significantly enhanced anti-tumor activity in both ibrutinib-sensitive and -resistant ABC-DLBCL cell lines. PI3K Regulates BCR-Dependent and BCR-Independent Activation of F-kB in ABC-DLBCL Activation of F-kB is a pathogenic hallmark of ABC-DLBCL (Compagno et al., 2009). To quantitate TFAIP3 effects on FkB, we established TMD-8, HBL-1, WSU-DLCL2, and U2932 cell lines with stably transfected F-kB-luciferase reporter. Copanlisib showed potent inhibitory effects on the F-kB reporter activities in all four cell lines with IC 50 and IC 90 values ranging from 4 to 26 nm and 24 to 262 nm, respectively (Figure 2A). Idelalisib and BYL-719 revealed anti-f-kb activity in the cell lines with low and high PI3Ka:PI3Kd expression, respectively, while ibrutinib exhibited potent IC 50 values in CD79 mut /MYD88 mut TMD-8 (2 nm) and HBL-1 (3 nm) cell lines. However, in contrast to copanlisib, ibrutinib even at 5 mm could not reach IC 90, suggesting that the inhibition of BTK is insufficient to completely block F-kB activation in CD79B mut /MYD88 mut TMD-8 and HBL-1 cell lines. In U2932, a cell line harboring an F-kB activating mutation of TFAIP3 downstream of BCR, surprisingly, ibrutinib potently blocked F-kB activation and achieved IC 50 and IC 90 at 1 and 7 nm, respectively, despite being unable to inhibit U2932 cell proliferation (Figures 1E and 2A; Campbell Cancer Cell 31, 64 78, January 9,

6 A B C D F E Figure 2. Regulation of uclear F-kB Activity by PI3K and BTK Inhibitors in ABC-DLBCL Cell Lines (A) Effect of PI3K and BTK inhibitors on nuclear activation of F-kB was assessed using cell lines with stably transfected F-kB-luciferase reporter constructs. Cells were treated with a series of dilutions of each compound in triplicate and luciferase activity was determined at 24 hr after treatment. IC 50 (Aa) and IC 90 (Ab) values were determined. *Greater than the maximum concentration tested. (B) Effect of PI3K and BTK inhibitors on F-kB target gene expression measured by qrt-pcr analysis of F-kB target gene expression in U2932 cells. (C) Effect of PI3K and BTK inhibitors on F-kB-regulated CCL4 production in TMD-8 and U2932 assessed using an ELISA assay at 24 hr after treatment. (D and E) Regulation of nuclear translocation of p65 F-kB by copanlisib, idelalisib, and ibrutinib in OCI-Ly3 (D) and TMD-8 cells (E). IHC staining with a primary antibody against p65 F-kB (red) and DAPI to visualize nuclear DA (blue) was performed at the indicated time points after treatment. (F) IC 50 isobolograms and combination indices (CIs) for idelalisib and BYL-719 combination in TMD-8 (Fa), HBL-1 (Fb), and WSU-DLCL2 (Fc) cell lines. See also Figure S2 and Table S3. 68 Cancer Cell 31, 64 78, January 9, 2017

7 A Figure 3. In vivo Anti-tumor Activity and Mechanism of Action of PI3K and BTK Inhibitors in Ibrutinib-Responsive CD79B mut / MYD88 mut ABC-DLBCL Tumor Models (A) SCID mice bearing TMD-8 or LY2298 PDX were treated with copanlisib (intravenously) and ibrutinib (orally) at the indicated doses and dosing schedules. Tumor volume was measured twice weekly and reported as the mean volume ± SEM (a and c). S, non-significant, *p % 0.05, **p % 0.01, and ***p % compared with vehicle. Relative tumor volume is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal (b and d). CR, complete response; PR, partial response; SD, stable disease; and PD, progressive disease. (B) Tumors from the LY2298 study were collected at the indicated time point after compound treatment and subjected to western blot analysis to evaluate BTK, MAPK, AKT, and F-kB pathway regulation. See also Figure S3. B et al., 2013; aylor et al., 2011). This result contradicts the previous explanation that activation of F-kB by TFAIP3 mut caused the resistance to ibrutinib in U2932. The inhibitory effects on FkB were further supported by the experimental data that ibrutinib treatment strongly suppressed F-kB target gene CCL4 and IL10 expression at the mra levels (Figure 2B) and CCL4 production at protein levels in U2932 cells as effectively as in ibrutinib-sensitive TMD-8 cells (Figure 2C). As a stable F-kB reporter was unavailable for OCI-Ly3 cells, we performed IHC staining to investigate nuclear activation of the p65 F-kB subunit. Copanlisib at 0.5 mm completely prevented p65 nuclear localization at 4 and 24 hr in both OCI-Ly3 and TMD-8 cells, while ibrutinib at 1 mm failed to inhibit (even slightly enhanced) nuclear p65 in OCI-Ly3 (Figure 2D), despite clear inhibition observed in TMD-8 cells at both 4 hr and 24 hr (Figure 2E). Idelalisib showed initial inhibition at 4 hr but no inhibition at 24 hr in both OCI-Ly3 and TMD-8 cells. The differential effects of copanlisib, idelalisib, and ibrutinib on p65 in OCI-Ly3 were further confirmed by analysis of F-kB-mediated transcrip- tion of IL10 and IL6 (Figure S2A). To demonstrate that both PI3Ka and PI3Kd are involved in the activation of nuclear F-kB, a combination study was performed. Indeed, in the TMD-8, HBL-1, and WSU-DLCL2 cell lines, combinations of idelalisib and BYL-719 led to synergistic inhibitory effects on F-kB-luciferase reporter activity with combination indices (CI) ranging from 0.18 to 0.38 (very strong synergy; Figure 2F). This conclusion was further confirmed by a synergistic inhibitory effect of BYL-719 and idelalisib on F-kBregulated transcription of CCL4, IL10, and IL6 (Figure S2B). Collectively, our data indicate that dual inhibition of PI3Ka and PI3Kd is required for effectively blocking F-kB activation mediated by BCR-dependent (CD79 mut ) and BCR-independent (e.g., MYD88 mut /CARD11 mut and TFAIP3 mut ) molecular alterations in ABC-DLBCL. PI3Ka/d Inhibition Showed Potent In Vivo Activity in CD79 mut /MYD88 mut ABC-DLBCL Models with a Distinct Molecular Mechanism In the CD79 mut /MYD88 mut TMD-8 xenograft tumor model in mice, treatment with copanlisib at 10 and 14 mg/kg every second day (Q2D) resulted in tumor growth inhibition (TGI) of 48% (p = 0.1) and 81% (p < 0.001), respectively (Figure 3Aa). Ibrutinib at 20 mg/kg (a dose corresponding to the exposure in humans at the maximum tolerated dose [MTD]) and 12 mg/kg led to TGI of 75% (p < 0.001) and 64% (p < 0.01), respectively, which is in line with previous findings (Staudt et al., 2011). o tumor regression was observed with copanlisib and ibrutinib (Figure 3Ab). To explore the concept of oncogenic shock and mimic the exposure profile of the intermittent schedule of copanlisib in the clinic, we tested a CD79 mut /MYD88 mut LY2298 PDX model with a 2-day Cancer Cell 31, 64 78, January 9,

8 on/5-day off (2On/5Off) schedule considering the shorter T 1/2 of copanlisib in mice (0.7 hr) than in humans (36 44 hr) (Liu et al., 2013a; Patnaik et al., 2016). The anti-tumor activity of ibrutinib and copanlisib was confirmed in the LY2298 PDX model with TGIs of 64.6% (p = 0.02) and 82.0% (p = 0.006), respectively (Figure 3Ac). In this study, ibrutinib treatment produced a partial response (PR) in 1/10 mice and copanlisib had 1/10 PR and 2/ 10 complete response (CR) (Figure 3Ad). To address the mechanisms of tumor growth inhibition in CD79 mut /MYD88 mut tumors, we investigated inhibition of the PI3K, MAPK, BTK, IKK/F-kB pathways by measuring the corresponding downstream signaling molecules. Treatment with copanlisib inhibited p-akt, p-ikba, and ciap1 (cellular inhibitor of apoptosis 1) at 3, 7, and 24 hr, and p-erk and caip2 at 7 and 24 hr in tumor samples from the LY2298 PDX model (Figure 3B). However, an increase in p-btk was observed at 3, 7, and 24 hr after copanlisib treatment. As a consequence of F-kB inhibition, a reduction of IL10 expression was observed upon copanlisib treatment (Figure S3). Treatment with ibrutinib transiently reduced p-btk, p-erk, and IL10 expression at 3 and/or 7 hr, however, it increased IL10, p-akt, and p-erk at 24 hr (Figures 3B and S3). These results suggested that rebound activation of BTK and AKT pathways upon PI3K and BTK inhibition, respectively, might be a mechanism limiting the PI3K and BTK inhibitors to show a robust and sustained response as monotherapy in CD79 mut /MYD88 mut DLBCL. PI3Ka/d Inhibition Demonstrated Potent In Vivo Activity in Ibrutinib-Resistant CARD11 mut and/or MYD88 mut DLBCL Models ABC-DLBCL patients with MYD88 mut /CD79 WT failed to respond to ibrutinib even though a high ORR was demonstrated in CD79B mut /MYD88 mut ABC-DLBCL (71%; Wilson et al., 2015). Based on the potent IC 90 observed with copanlisib in the in vitro F-kB reporter assay, we hypothesized that in contrast to ibrutinib, copanlisib could inhibit MYD88 mut -mediated activation of F-kB. To test this, we selected the LY0257 PDX model bearing MYD88 mut but wild-type CD79A/B. Strikingly, treatment with copanlisib at 14 mg/kg with a 2On/5Off schedule led to a mean TGI of 89.4% (p < versus the vehicle group; Figure 4Aa), while the ibrutinib treatment group exhibited constant tumor growth with a mean TGI of 41.7% on day 16. On day 21, 4/10 mice reached PR and 1/10 mice showed complete tumor growth control in the copanlisib-treated group (Figure 4Ab). This dosing regimen was well tolerated with body weight loss <4% (Figure S4A). Inhibition of the F-kB pathway was demonstrated by reduction of IL10 expression (Figure 4Ba) and ciap1/ 2 at the protein level (Figure 4Ca) in tumors 3 and/or 24 hr after copanlisib treatment in vivo, while ibrutinib only showed transient IL10 inhibition at 3 hr but not at 24 hr, which was confirmed by the p-stat3 levels in the corresponding tumor lysates (Figures 4Ba and 4Ca). In addition, sensitivity to PI3K inhibition was also associated with sustained suppression of p-akt in the LY0257 tumor model (Figure 4Ca). Interestingly, similar to what was observed in the two MYD88 mut /CD79 mut tumor models (Figures 3B and 4B), rebound activation of BTK was also found in the MYD88 mut LY0257 model after copanlisib treatment (Figure 4Ca). We also examined the effect of copanlisib and ibrutinib in the OCI-Ly3 subcutaneous xenograft model. Consistent with the in vitro observation, copanlisib displayed moderate but significant anti-tumor activity with TGI of 62.0% (p < 0.05; Figures 4Ac and 4Ad), while ibrutinib had limited activity (TGI = 19.2%). Furthermore, copanlisib treatment exhibited substantial inhibition of F-kB-mediated transcription of IL10 at 1 and 7 hr after the last dosing, while only a moderate effect at 1 hr and no effect at 7 and 24 hr were observed in the ibrutinib-treated group (Figure 4Bb). Furthermore, high p-akt expression in OCI-Ly3 tumors was completely inhibited by copanlisib but not by ibrutinib (Figure 4Cb). otably, we observed substantial p-erk and p-btk levels even in the copanlisib group, suggesting that activation of the MAPK and BTK pathways in OCI-Ly3 may counteract the anti-tumor efficacy of copanlisib. PI3Ka/d Inhibition-Based Combination Strategy for the Treatment of ABC-DLBCL One plausible mechanism for the inability of ibrutinib or copanlisib alone to induce a sustained tumor response might be the rebound activation of AKT and BTK, respectively (Figures 3B and 4C). We therefore conducted an in vitro evaluation of copanlisib in combination with ibrutinib using isobologram/ci assessment followed by mechanism analysis. Initial data published by us (Liu et al., 2013b) and others (Griner et al., 2014) suggest a synergistic combination effect of PI3K and BTK inhibitors in some ABC-DLBCL cell lines. However, here we show that copanlisib and ibrutinib combination is not always synergistic. In the ibrutinib-sensitive (IC 50 below 400 nm) TMD-8, HBL-1, and WSU-DLCL2 cell lines, combination of copanlisib and ibrutinib exhibited strong synergistic effects with regard to both IC 50 and/or IC 90 (Figures 5Aa, 5Ab, and S5A). On the other hand, U2932, a cell line sensitive to ibrutinib-mediated F-kB suppression, but resistant to ibrutinib in a proliferation assay, displayed a synergistic anti-proliferative effect measured by IC 50, but no enhanced effect on IC 90 (Figures 5Ac and 5Ad). More strikingly, in ibrutinib-resistant CARD11 mut OCI-Ly3 cells, the combination of copanlisib with ibrutinib generated strong antagonistic effects with CIs ranging from 1.49 to 2.75 (Figure 5Ae). The synergistic anti-proliferative activity of copanlisib and ibrutinib in TMD-8 was supported by the strongly enhanced inhibitory effect on F-kB (CI, on IC 90 )(Figure 5Bb) and p-erk together with the potent suppression of p-akt by copanlisib and p-btk by ibrutinib (Figure 5Bc). Consequently, the combination of the two agents led to induction of apoptosis in TMD-8 cells (Figure 5Bc). In ibrutinib-insensitive U2932 cells, a synergistic anti-proliferative effect of copanlisib and ibrutinib was observed with regard to IC 50 but not IC 90 (Figures 5Ac and 5Ad). Compared with TMD- 8 cells, the expression of p-akt was much higher in U2932 cells, which might be associated with the resistance to ibrutinib. otably, although complete inhibition of F-kB, p-akt, and p-erk was achieved by combination of copanlisib and ibrutinib (Figure 5C), it failed to lower the concentration of copanlisib and ibrutinib required to reach IC 90 in the proliferation assay (Figure 5Ad). Further investigation revealed that in comparison with other ABC-DLBCL cell lines, U2932 cells expressed remarkably higher levels of Mcl-1 (Figure S5B), which may provide survival signaling against the combination treatment of copanlisib and ibrutinib. 70 Cancer Cell 31, 64 78, January 9, 2017

9 A B C Figure 4. In vivo Anti-tumor Activity and Mechanism of Action of PI3K and BTK Inhibitors in Ibrutinib-Resistant MYD88 mut and/or CARD11 mut ABC-DLBCL Xenograft Tumor Models (A) SCID mice bearing LY0257 PDX (MYD88 mut ) and OCI-Ly3 (MYD88 mut /CARD11 mut ) xenografts were treated with copanlisib (intravenously) and ibrutinib (orally) at the indicated doses and dosing schedules. Tumor volume was measured twice weekly and reported as the mean volume ± SEM (a and c). Relative tumor volume (b and d) is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal. CR, complete tumor response; PR, partial response; SD, stable disease; and PD, progressive disease. *p % 0.05, ***p % compared with vehicle. (B) F-kB activity was assessed by IL10 gene expression analysis in LY0257 (a) and OCI-Ly3 tumors (b) by RT-PCR. (C) Western blot analysis of BTK, AKT, MAPK, and F-kB pathway regulation in LY0257 tumors (a) 3 or 24 hr after treatment and in OCI-Ly3 tumors (b) 1, 7, or 24 hr after treatment with copanlisib or ibrutinib. See also Figure S4. Cancer Cell 31, 64 78, January 9,

10 Despite the single-agent activity of copanlisib in inhibiting FkB and tumor growth in OCI-Ly3 in vitro and in vivo (Figures 1E, 2D, 4A, and 4B), combining copanlisib with ibrutinib generated unwanted antagonistic effects (Figure 5Ae). Further analysis of oncogenic signaling pathways revealed that neither copanlisib nor ibrutinib could inhibit p-erk in OCI-Ly3 cells and the combination even led to an increase of p-erk level (Figure 5Dc). This result was in contrast to the synergistic inhibitory effect on p-erk in TMD-8 cells (Figure 5Bc), which may explain the antagonistic combination of copanlisib and ibrutinib. To evaluate the role of p-erk in promoting OCI-Ly3 proliferation and survival, we tested the combination effect of copanlisib and the MEK inhibitor refametinib (BAY ) in OCI-Ly3 cells. As depicted in Figures 5Da and 5Db, isobologram analysis of IC 50 and IC 90 revealed a very strong synergistic combination of copanlisib and refametinib (CI, for IC 50 and for IC 90 ). This result confirmed that, in addition to the inhibition of F-kB and p-akt by copanlisib, inhibition of the MAPK pathway may be required to completely block OCI-Ly3 survival. Combination of Copanlisib and Ibrutinib Resulted in Complete Tumor Remission in CD79 mut /MYD88 mut DLBCL Xenograft Models in Mice The combination of copanlisib and ibrutinib was first tested in the TMD-8 model in C.B17 SCID mice with two schedules. Dosing copanlisib (14 mg/kg) with a 2On/5Off schedule displayed a slightly weaker activity compared with a Q2D regimen (TGI = 63.3% versus 82.8%; Figure 6Aa) as monotherapy. When combining with ibrutinib, both schedules achieved a 100% response rate with CR observed in 5/8 mice in the 2On/5Off group and all 5 mice with the Q2D schedule. The combination effect was significant versus ibrutinib monotherapy (p < 0.001). However, copanlisib dosed with the 2On/5Off schedule in combination with ibrutinib was much better tolerated compared with copanlisib with the Q2D schedule (body weight change 3.2% versus 9.1%, respectively; Figure S6). The combination of copanlisib and ibrutinib was further tested in the LY2298 PDX model (passage 6) in OD/SCID mice. Copanlisib with the 2On/5Off schedule was very well tolerated with no body weight loss in any treatment group during an 81-day study period (Figure 6Ba). In this study, treatment was conducted in two to three phases to address the sensitivity to copanlisib and/or ibrutinib treatment in naive, refractory, reoccurring, and relapsed tumors. In the first phase of treatment (day 0 18), mice in groups 1, 2, 3, and 4 (8 mice/group) were treated with vehicle, copanlisib, ibrutinib, and copanlisib plus ibrutinib, respectively. Copanlisib induced tumor regression with 6/8 CRs on day 18 (Figure 6Bc) and 2/8 animals did not show tumor shrinkage (Figure 6Bd), while ibrutnib resulted in delayed tumor growth with an average TGI of 56.4%. The combination of copanlisib and ibrutinib resulted in faster onset of CRs (7/8 on day 7 and 8/8 on day 14) compared with copanlisib alone (4/8 on day 7 and 5/8 on day 14). As treatment with ibrutinib did not reach tumor regression on day 18, copanlisib was added in the second treatment phase (day 19 49) and induced 3/8 CRs after 1 week and 7/8 CRs after 2 weeks of combination treatment. Surprisingly, tumors initially refractory to copanlisib (group 2B) did not respond and eventually became resistant to the combination therapy (Figure 6Bd). To address tumor recurrence, treatment was stopped once CR was achieved for 1 2 weeks and tumor regrowth was monitored. Interestingly, tumor recurrence was observed in 1 week after stopping treatment in mice that received monotherapy of copanlisib (Figure 6Bc) or initially ibrutinib and then combination treatment (Figure 6Be), while a significantly longer time (>5 weeks) was required for tumor recurrence in mice initially treated with combination therapy. In addition, tumor recurrence from combination treatment still responded to combination therapy with 6/8 CRs within a week (Figure 6Bf, day 70 77). Furthermore, 50% of mice (3/6) with reoccurring tumors in group 2A still responded to the second copanlisib treatment, while the other three mice relapsed on day Strikingly, tumors relapsed from copanlisib monotherapy were still sensitive (3/3 CRs) to the copanlisib and ibrutinib combination treatment (Figure 6Bc, day 49 60). Together, these data support a clinical study using copanlisib in combination with ibrutinib in CD79 mut ibrutinib-naive, refractory, or relapsed ABC-DLBCL. DISCUSSIO Class I PI3K isoforms (PI3Ka, PI3Kb, PI3Kg, and PI3Kd) have distinct expression profiles and functions in oncogenic signaling (Rommel et al., 2007; So and Fruman, 2012). Pharmacological inhibition of the PI3K/mTOR pathway has been shown to suppress lymphoma growth and survival in preclinical tumor models (Dasgupta et al., 2005; Kloo et al., 2011; Uddin et al., 2006), but the relative expression and functional importance of each PI3K isoform in different lymphoma subtypes as well as the downstream signaling beyond the PI3K/AKT/mTOR pathway are largely unknown. The recent approval of idelalisib validated the role of PI3Kd in FL, CLL, and SLL (Gopal et al., 2014), but the effect of idelalisib in DLBCL has been disappointing. By comparing PI3K isoform expression in FL and DLBCL, we proposed a rationale of testing a PI3Ka/d inhibitor in ABC-DLBCL. Firstly, the incidence of PI3Ka high was dramatically increased in ABC-DLBCL (57%) compared with FL (18%). Secondly, the prevalence of PTE-loss (inducing PI3Kb activation) was lower in ABC-DLBCL (14%) compared with GCB-DLBCL (70%). The five cell lines selected for functional characterization of PI3Ka/d isoforms represent the key molecular features of ABC- DLBCL, including a differential PI3Kd:PI3Ka expression ratio. Using both IC 50 and IC 90 in a set of quantitative assessments, the functional role of PI3Ka expression in causing intrinsic resistance to PI3Kd-selective inhibition in ABC-DLBCL was elucidated. (1) The inhibitory activity of idelalisib and BYL-719 on F-kB and proliferation correlated with high and low PI3Kd:PI3Ka expression ratios, respectively. (2) In contrast to PI3Ka/d dual inhibition by copanlisib, idelalisib revealed only moderate to no or incomplete anti-proliferative activity (IC 90 >5 mm) in the five cell lines tested, despite idelalisib and copanlisib showing comparable cellular potency against PI3Kd. Furthermore, the combination of BYL-719 and idelalisib demonstrated strong synergistic effects in suppressing F-kB nuclear activation and p-akt and cell survival. In addition, rebound activation of F-kB was observed in both TMD-8 and OCI-Ly3 cells at 24 hr after idelalisib treatment, but this is not the case for copanlisib or ibrutinib. Together, our data provide a molecular mechanism underlining the intrinsic and acquired resistance to PI3Kd selective inhibition in ABC-DLBCL. In addition to DLBCL, 72 Cancer Cell 31, 64 78, January 9, 2017

11 A B C D Figure 5. In Vitro Combination Effects of PI3K Inhibitor Copanlisib and BTK Inhibitor Ibrutinib in ABC-DLBCL Models (A) IC 50 and IC 90 isobolograms of copanlisib and ibrutinib combination were generated using a 72-hr CellTiter-Glo proliferation assay. Combination effects were assessed using IC 50 and/or IC 90 isobolograms for CD79B mut /MYD88 mut TMD-8 (a and b) and TAK1 mut /TFAIP3 mut U2932 (c and d) cells and antagonistic combination in MYD88 mut /CARD11 mut OCI-Ly3 cells (e and f). (legend continued on next page) Cancer Cell 31, 64 78, January 9,

12 an increase in PI3Ka expression was also observed in late-disease-stage FL patients and correlated with a high FLIPI risk factor. In line with our findings, increased protein expression of PI3Ka was observed in MCL after relapse (Iyengar et al., 2013). Together these results suggest that dual inhibition of PI3Ka/ d might be a more effective therapeutic approach to overcome intrinsic and acquired resistance in both ihl and ahl. Indeed, a preclinical study comparing PI3Kd inhibition by idelalisib, PI3Kd/g inhibition by IPI-145, and PI3Ka/d inhibition by copanlisib in primary tumor cells from CLL patients revealed that copanlisib is the most effective approach to inhibit CLL cell survival (Gockeritz et al., 2015). The mutations that lead to the constitutive activation of F-kB in ABC-DLBCL are CD79A/B mut, CARD11 mut, TFAIP3 mut, TAK1 mut, and MYD88 mut (Compagno et al., 2009). Several targeted agents, e.g. inhibitors targeting SYK, BTK, and PKC, are particularly active in CD79A/B mut ABC-DLBCL cell lines but incapable of blocking tumor cell proliferation in DLBCL cell lines with mutations in CARD11, TFAIP3, and TAK1 (aylor et al., 2011). Clinical testing of ibrutinib confirmed the preclinical data and further identified an additional patient population with CD79 WT / MYD88 mut resistant to BTK inhibition (Wilson et al., 2015). The potential involvement of class I PI3K isoforms in the regulation of F-kB was indicated in a subset of BCR-addicted TMD-8 and HBL-1, but was not the case in CARD11 mut OCI-Ly3 and TFAIP3 mut U2932 cell lines in Kloo et al. (2011). In this study, we found that dual inhibition of PI3Ka/d, e.g., by copanlisib, could attenuate F-kB activation not only in BCR-addicted (e.g., CD79 mut TMD-8 and HBL-1) but also in BCR-independent (e.g., CARD11 mut, TAK1 mut /TFAIP3 mut, or CD79 WT /MYD88 mut ) ABC-DLBCL tumor models (OCI-Ly3, U2932, and LY0257) in an AKT-independent manner (Table S2). Modulation of F-kB activity was assessed by various in vitro and in vivo assays, including cell lines with stably transfected F-kB reporter constructs, transcriptional regulation of F-kB target genes, F-kB-mediated cytokine/chemokine production, and nuclear translocation of F-kB. In line with the previous publication (Kloo et al., 2011), copanlisib potently inhibited p-ikba in CD79B mut LY2298. On the other hand, p-ikba could not be detected in a CD79 WT /MYD88 mut LY0257 PDX model (data not shown), suggesting that an alternative signaling pathway may mediate F-kB activation. Interestingly, inhibition of F-kB by copanlisib appeared to be associated with inhibition of ciap1 and ciap2 in both BCR-dependent CD79B mut /MYD88 mut LY2298 and BCR-independent CD79 WT /MYD88 mut LY0257 PDX tumor models. These results provide an initial molecular link between PI3K inhibition and suppression of F-kB in ABC- DLBCL. Regulation of ciap1/2 expression by PI3K inhibitor has been reported before in tumor-necrosis-factor alpha-stimulated lung (Petersen et al., 2010) and breast tumor cell lines (Liu et al., 2006). We demonstrated here that a highly selective PI3K inhibitor could potently inhibit F-kB activity in ABC-DLBCL tumor models covering the key genetic features for F-kB activation, inviting more dedicated research to elucidate detailed molecular mechanisms. BCR and F-kB activation have been considered as important drivers of tumor growth and survival in ABC-DLBCL. Our in vitro and in vivo analyses indicate that the PI3K/AKT and MEK/ERK signaling pathways are also important and even more prominent than the BCR/F-kB pathway in a subset of tumor models, such as LY0257, OCI-Ly3, and U2932. In several previous publications, inhibitors targeting the BCR/BTK/F-kB pathway, e.g., inhibitors of SYK (R406), BTK (ibrutinib), PKC (sotrastaurin), BHA536 (enzastaurin), MALT1 (MI2), IKKb (AF700, ML120B), as well as PI3Kd (idelalisib) and PI3Kg (duvelisib), did not show significant anti-proliferative activity in U2932, which has been attributed to downstream F-kB activation by TAK1 mut and TFAIP3 mut (Campbell et al., 2013; Fontan et al., 2012; aylor et al., 2011). In contrast, we found that ibrutinib actually exhibited even more potent activity (3- to 4-fold) in blocking F-kB activation (IC 50 /IC 90 = 1 nm/7 nm) than copanlisib (IC 50 /IC 90 = 4 nm/ 24 nm) in the F-kB reporter assay. Despite potent inhibition of F-kB, ibrutinib was almost inactive (IC 50 = 3.5 mm) in blocking cell proliferation, while copanlisib exhibited potent anti-proliferative activity (IC 50 = 18 nm). This result implicates a predominant activation of the PI3K pathway in U2932 cells. Indeed, we found that p-akt levels are much higher in U2932 compared with BCRaddicted TMD-8 cells. Hence, PI3K-mediated p-akt, rather than F-kB, may be a key driver promoting U2932 cell proliferation. It remains to be elucidated to what extent the mutations in TFAIP3 and TAK1 contribute to the nuclear activation of F-kB and how ibrutinib effectively regulates F-kB activity in U2932 cells. Overall, compared with ibrutinib, copanlisib demonstrated a more potent and broad anti-tumor profile in ABC-DLBCL models in vitro and in vivo through inhibiting p-akt induced by BCR/SYK/PI3K, CD19/PI3K, and/or CD40/ PI3K (Hojer et al., 2014) and nuclear activation of F-kB induced by both BCR/BTK-dependent and BCR/BTK-independent mechanisms. Despite the clear anti-tumor activity of PI3Ka/d inhibition by copanlisib in multiple tumor models, several aspects promote combination therapies to achieve a sustained tumor response. One important finding of this study is that PI3K inhibition caused rebound activation of BTK in both ibrutinib-sensitive (TMD-8 and LY2298) and ibrutinib-resistant (LY0257 and OCI-Ly3) ABC- DLBCL models as well as selective PI3Kd inhibition by idelalisib-induced (but not PI3Ka inhibition by copanlisib) rebound activation of F-kB, indicating a potential synergy by simultaneous inhibition of PI3Ka and BTK. Interestingly, the combination of copanlisib and ibrutinib produced different outcomes in vitro. In ibrutinib-sensitive tumor models, synergistic inhibition of F-kB and p-erk was observed, which indeed resulted in enhanced tumor cell apoptosis in vitro and sustained complete tumor response in vivo. On the other hand, in ibrutinib-resistant tumor models, like OCI-Ly3, the combination led to antagonistic anti-tumor effects. One plausible mechanism underlying (B and C) Mechanistic combination effects on inhibition of F-kB were measured using a luciferase reporter assay (a and b), and inhibition of p-akt, p-erk, and p-btk was assessed by western blot (c) and in TMD-8 (B) and U2932 (C) cells, as well as the functional consequence on tumor cell survival by measuring apoptotic protein cleaved PARP (Bc) and Mcl-1 (Cc). (D) Functional and mechanistic combination effects in OCI-Ly3 cells were assessed by combination of copanlisib with the MEK inhibitor refametinib in a CTG assay (a and b) and by western blot analysis of p-akt, p-btk, and p-erk (c). See also Figure S5. 74 Cancer Cell 31, 64 78, January 9, 2017

13 A B Figure 6. In Vivo Combination Effects of the PI3K Inhibitor Copanlisib and the BTK Inhibitor Ibrutinib (A) SCID mice bearing TMD-8 tumors were treated with copanlisib (14 mg/kg, intravenously), ibrutinib (20 mg/kg, orally), and their combination at the indicated dosing schedules shown in the legend). Tumor volume was measured twice weekly and reported as the mean ± SEM (a). ***p % compared with vehicle and ### p % compared with ibrutinib monotherapy. Relative tumor volume (b) is defined as the percentage of the final tumor volume versus the initial tumor volume (at the time point of starting treatment) of each individual animal. CR, complete tumor response; PR, partial response; SD, stable disease; and PD, progressive disease. Ibrut refers to ibrutinib and copan refers to copanlisib. (B) SCID mice bearing LY2298 PDX were treated with copanlisib (14 mg/kg, intravenously), ibrutinib (20 mg/kg orally), and their combination at the indicated dosing schedule (red bar indicates copanlisib schedule and gray bar indicates ibrutinib schedule) in the treatment groups 2A, 2B, 3, and 4 (c, d, e, and f, respectively). Relative body weight change during the treatment was calculated by comparison with the initial body weight on day 0 (a). Tumor size was reported as the mean ± SEM during the treatment (b f). Models resistant to combination therapy are marked as x. Complete response (CR) rate during the treatment is indicated at the bottom of each graph. See also Figure S6. Cancer Cell 31, 64 78, January 9,

14 Figure 7. Targeting PI3Ka/d for the Treatment of ABC-DLBCL (A) PI3K-mediated survival signaling pathways in ABC-DLBCL. In addition to activating the AKT pathway, PI3Ka/d, the two isoforms predominantly expressed in ABC-DLBCL, also regulate BCRdependent and -independent activation of the FkB pathway via ciaps and p-ikb. The STAT3 signaling pathway is indirectly modulated by PI3K via F-kB target genes IL10 and IL6. Furthermore, negative crosstalk between PI3K and BTK results in a rebound activation of p-btk and p-akt upon inhibition of PI3K and BTK, respectively. (B) Combination of PI3Ka/d and BTK inhibitors is proposed for the treatment of ABC-DLBCL with CD79 mut or WT CD79 and the F-kB pathway, while PI3Ka/d inhibition in combination with SoC, e.g. R-CHOP, is proposed for the treatment of ABC-DLBCL with BCR-independent activation of F-kB. synergistic versus antagonistic effects could be the combination outcomes on the regulation of p-erk levels. This hypothesis was supported by the synergistic combination of copanlisib with the MEK inhibitor refametinib in OCI-Ly3. Tolerability has been a concern for PI3K and BTK inhibitor combination therapy. In the clinic, copanlisib dosed intravenously once weekly showed lower incidence and severity of gastrointestinal (e.g., diarrhea, colitis) and liver toxicity compared with continuously dosed oral PI3K inhibitors (Patnaik et al., 2016). Therefore, we tested two schedules of copanlisib, Q2D to get an overall exposure coverage and 2On/5Off to mimic clinical intermittent exposure, in combination with ibrutinib dosed daily. Consistent with the better safety profile observed with intermittent schedule in the clinic, the 2On/5Off schedule was well tolerated as a single agent and in combination with ibrutinib in the TMD-8 model in C.B17 SCID mice and in the LY2298 PDX model in OD/SCID mice. More importantly, the combination of copanlisib and ibrutinib led to the following therapeutic benefits, which are relevant in the clinical setting: (1) faster onset and higher rate of complete tumor response; (2) significantly prolonged time to tumor recurrence (>5 weeks in the combination group versus 1 week in the copanlisib treatment group 2); (3) active in ibrutinib refractory/relapsed tumors; (4) active in recurring and relapsed tumors from copanlisib treatment. The anti-tumor activity of copanlisib in combination with ibrutinib in the CD79 mut /MYD88 mut TMD-8 and LY2298 PDX models is, to our knowledge, the most effective ibrutinib combination published so far (e.g., Ceribelli et al., 2014). In summary, dual inhibition of PI3Ka/ d led to potent inhibition of two important survival signaling pathways: the PI3K/ AKT and F-kB pathways in both BCR-dependent and -independent ABC- DLBCL (Figure 7A). PI3Ka/d inhibition by copanlisib demonstrated promising antitumor activities in vitro and in vivo in multiple tumor models, representing the key features of ABC-DLBCL with a well-tolerated intermittent dosing schedule. Therefore, a molecularly based clinical exploration of copanlisib in combination with standard of care chemotherapy, ibrutinib, or other targeted therapies is warranted as a promising therapeutic approach for the treatment ABC-DLBCL (Figure 7B). EXPERIMETAL PROCEDURES IHC Analysis of PI3K Isoform Expression in Patient Samples Archived formalin-fixed paraffin-embedded samples of FL and DLBCL collected between 2005 and 2013 (Advanced Molecular Pathology Lab, Singapore Health Services Pte Ltd) were randomly selected and constructed into tissue microarrays. Antigenicity of tissue cores was confirmed to be wellpreserved by Ki67 IHC (Ki67 in at least 10% of neoplastic cells). Finally, 45 DLBCL and 45 FL cases were selected for this study. IHC staining was performed using AMPL s ER1 protocol. A qualified pathology associate performed subsequent blinded evaluation. A positive score means that there was expression in at least 1% of tumor cells. The intensity was graded as 0, negative staining; 1+, mild expression; 2+, moderate expression; 3+, strong expression. Inhibition of Cell Proliferation and PI3K Pathway Activity The effect of testing PI3K, BTK, and MEK inhibitors on tumor cell growth was determined using cell viability assays as described previously (Liu et al., 76 Cancer Cell 31, 64 78, January 9, 2017