Compared with follicular lymphoma, high PI3Kα expression was more prevalent in diffuse large B cell lymphoma (DLBCL), although both tumor types expressed substantial PI3Kδ. Simultaneous inhibition of PI3Kα and PI3Kδ dramatically enhanced the anti-tumor profile in ABC-DLBCL models compared with selective inhibition of PI3Kδ, PI3Kα, or BTK. The anti-tumor activity was associated with suppression of p-AKT and a mechanism of blocking nuclear factor-κB activation driven by CD79, CARD11, TNFAIP3, or MYD88. Inhibition of PI3Kα/δ resulted in tumor regression in an ibrutinib-resistant CD79B/MYD88 patient-derived ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79B-ABC-DLBCL to show a robust response to PI3K and BTK inhibitor monotherapies. A combination of ibrutinib with the PI3Kα/δ inhibitor copanlisib produced a sustained complete response in vivo in CD79B/MYD88 ABC-DLBCL models.

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo fatty acid synthesis, and its ACC1 isoform is overexpressed in pancreatic and various other cancers. The activity of many oncogenic signaling molecules, including WNT and Hedgehog (HH), is post-translationally modified by lipidation. Here, we report that inhibition of ACC by a small molecule inhibitor, BAY ACC002, blocked WNT3A lipidation, secretion, and signaling. In pancreatic cancer cells, where WNT and HH are key oncogenic drivers, ACC inhibition simultaneously suppressed WNT and HH signaling, and led to anti-proliferative effects. Treatment with ACC inhibitors blocked tumor growth and converted the poorly differentiated histological phenotype to epithelial phenotype in multiple cell line-based and patient-derived pancreatic cancer xenograft models. Together, our data highlight the potential utility of ACC inhibitors for pancreatic cancer treatment, and provide novel insight into the link between upregulated de novo fatty acid synthesis in cancer cells, protein lipidation, and oncogenic signaling.

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Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two of the most common Non-Hodgkin Lymphoma (NHL) wordwide. Although the introduction of anti-CD20 monoclonal antibodies has improved the outcome of patients with follicular lymphoma, a curative treatment or an improved treatment strategy for relapsed FL are still to be developed. DLBCL is an incurable, aggressive subtype of NHL with a high unmet medical need for effective therapeutics. Activation of the PI3K-AKT pathway by B cell receptor signaling and its role in the pathogenesis of FL and DLBCL have been highlighted in a number of studies, however, the relative importance of PI3K isoforms and effective application of PI3K inhibitors for the treatment of FL and DLBCL have not been fully addressed. To answer this question, we selected and characterized a panel of cell lines representing major subtypes (e.g. ABC and GCB) and frequent mutations (e.g. CD79, Bcl2, MyD88, CARD11, or EZH2) in FL and DLBCL. Analyzing the expression of PI3K isoforms indicated that not only PI3Kδ, an isoform known to be enriched in lymphocytes, but also PI3Kα is highly expressed in DLBCL and a subset of FL cell lines. Sensitivity profiling of the PI3Kα/δ inhibitor BAY 80-6946 (biochemical IC50=0.5 nM and 0.7 nM against PI3Kα and PI3Kδ, respectively) and the PI3Kδ-selective inhibitor CAL-101 confirmed that inhibition PI3Kα/δ is more effective than inhibition of PI3Kδ, particularly PI3Kα/δ inhibition showed a broader anti-tumor spectrum in the cell line panel. For example, unlike CAL-101, BAY 80-6946 revealed potent anti-tumor activity in NFκB activating MyD88 or CARD11 mutated DLBCL cell lines both in vitro and in vivo. Further analysis of mutation status and pathway inhibition discovered an unknown PI3K isoform-dependent crosstalk to the NFκB pathway in DLBCL. The detailed molecular mechanism and the strategy of developing PI3Kα/δ inhibitor as a single agent and rationale-based combination in FL and DLBCL will be discussed. Taken together, these findings provide further insights into the mechanism of action of BAY 80-6946 and support ongoing Phase I studies in FL and DLBCL patients (Lotze et al., J Clin Oncol 30, 2012, suppl, abstr 3019, data to be updated in the presentation). Citation Format: Ningshu Liu, Katja Haike, Juliane Paul, Isabelle Genvresse, Antje M. Wengner, Dirk Laurent, Damian O'Connell, Dominik Mumberg, Karl Ziegelbauer. PI3Kα/δ inhibition has greater efficacy compared to PI3Kδ-selective inhibition in NHL with activated NFkB pathway . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4115. doi:10.1158/1538-7445.AM2013-4115

Introduction: ABC-DLBCL is a subtype of DLBCL with less favorable clinical outcomes to the standard of care (SoC) therapies. Constitutive activation of NF-κB by various genetic alterations in ABC-DLBCL has been identified as one of the key mechanisms driving chemotherapy resistance. Inhibition of B cell receptor (BCR) signaling with BTK (Bruton's tyrosine kinase) inhibitor ibrutinib demonstrated encouraging clinical responses in ABC-DLBCL. However, patients with CD79wt/MyD88mut, or CARD11mut did not respond to ibrutinib, indicating the need for new therapies targeting ibrutinib refractory ABC-DLBCL. Recent approval of PI3Kδ selective inhibitor idelalisib for the treatment of indolent NHL (iNHL) as monotherapy highlighted selective inhibition of PI3Kδ as an effective therapeutic strategy. However, idelalisib did not show clinical activity in DLBCL in a Phase I expansion cohort study. Here we report immunohistochemical (IHC) analysis of clinical tumor samples from follicular lymphoma (FL) and DLBCL patients and a series of in vitro and in vivo mechanistic and functional studies to explore the importance of PI3K isoforms in regulating key signaling pathways in ABC-DLBCL. Methods: Expression of PI3K isoforms and PTEN was assessed by IHC and western blot from a panel of 45 FL and 45 DLBCL primary tumors. Effects of PI3K inhibitors (PI3Kδ-selective inhibitor idelalisib, PI3Kα-selective inhibitor BLY-719, PI3Kβ-selective inhibitor TGX-221, predominant PI3Kα/δ inhibitor copanlisib and BTK inhibitor ibrutinib) on nuclear NF-κB activation were determined using stable cell lines expressing NF-κB-luciferase reporter (obtained by lentiviral infection), IHC staining of p65 NF-κB, expression of CCL4, IL-6, and IL-10 by RT-PCR and protein production by ELISA assays. In vitro and in vivo mechanisms of action were addressed by assessing the activities of the key survival signaling pathways. In vitro and in vivo anti-tumor activities were investigated using cell lines and patient derived xenograft ABC-DLBCL models representing the key molecular features of BCR-dependent and independent ABC-DLBCL. Results: Although PI3Kδ was predominantly expressed in both FL and DLBCL, high PI3Kα expression was more prevalent in DLBCL (60% vs 18%), a patient population resistant to PI3Kδ-selective inhibition in the clinic. Simultaneous inhibition of PI3Kα and PI3Kδ with BYL-719 plus idelalisib or copanlisib alone dramatically enhanced anti-tumor profile in ABC-DLBCL models compared to selective inhibition of PI3Kδ, PI3Kα or BTK. The anti-tumor activity was associated with suppression of p-AKT and a novel mechanism of blocking NF-κB activation driven by CD79mut, CARD11mut, A20mut or MyD88mutin vitro and in vivo. Suppression of NF-kB activation by PI3K inhibition is independent from AKT, but involves a novel mechanism of modulating c-IAP expression. Inhibition of PI3Kα/δ resulted in complete tumor regression in an ibrutinib-resistant MyD88mut-LY0257 patient-derived (PDx) ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79mut ABC-DLBCL to show robust response to PI3K and BTK inhibitors, respectively, as single agents in vivo. Combination of ibrutinib with PI3Kα/δ inhibitor copanlisib dosed intermittently iv was well tolerated and produced complete tumor regression in CD79Bmut TMD-8 cell line and Ly2298 PDx ABC-DLBCL models. Conclusions: High expression of PI3Kα in addition to PI3Kδ in ABC-DLBCL is associated with intrinsic resistance to PI3Kδ selective inhibition. Simultaneous inhibition of PI3Kα/δ by copanlisib modulates not only the PI3K/AKT pathway but also BCR-dependent and independent activation of nuclear NF-κB via a novel AKT-independent mechanism, indicating a promising utility for the treatment of clinically relevant ibrutinib-resistant ABC-DLBCL patients with CD79wt/MyD88mut, A20mut, or CARD11mut tumor genotypes. Combination of PI3Kα/δ and BTK inhibitors demonstrated promising potential for ibrutinib-responsive ABC-DLBCL to achieve complete tumor regression by blocking rebound activation of BTK and AKT. Thus, our findings presented here provide additional insights on intrinsic and acquired resistance mechanisms to selective PI3Kδ and BTK inhibitors and provide rationale for clinical development of PI3K inhibitors with specific isoform profile in combination for the treatment of ABC-DLBCL. Disclosures Paul: Bayer AG: Employment. Sojoun:Bayer AG: Employment. Wengner:Bayer AG: Employment. Zitzmann-Kolbe:Bayer AG: Employment. Sturz:Bayer AG: Employment. Haike:Bayer AG: Employment. Martin:Bayer AG: Employment. Mumberg:Bayer AG: Employment. Ziegelbauer:Bayer AG: Employment. Liu:Bayer AG: Employment.