Dual targeting non-overlapping epitopes in HER2 domain IV substantially enhanced HER2/HER2 homodimers and HER2/EGFR heterodimers internalization leading to potent antitumor activity in HER2-positive human gastric cancer.

BACKGROUND: Trastuzumab and pertuzumab combination has been approved for the treatment of patients with HER2-positive metastatic breast cancer. However, trastuzumab and pertuzumab combination did not show improvement in overall survival in patients with HER2-positive metastatic gastric cancer. METHODS: We developed a new HER2-targeted monoclonal antibody, HLX22, targeting HER2 subdomain IV as trastuzumab but with non-overlapping epitopes. We examined the antitumor effects of this novel HER2-antibody in gastric cell lines and cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. RESULTS: HLX22 in combination with HLX02 (trastuzumab biosimilar) induced enhancement of HER2/HER2 homodimers and HER2/EGFR heterodimers internalization, which ultimately led to the reduction in signal transductions involving STAT3, P70 S6, and AKT; gene expressions of FGF-FGFR-PI3K-MTOR, EGF-EGFR-RAS, TGF-ß-SMAD, PLCG and cell cycle progression related pathways that favor tumor development, proliferation, progression, migration and survival in gastric cancer cell line NCI-N87 were also reduced. These differing but complementary actions contributed to the synergistic antitumor efficacy of the HLX22 and HLX02 combination in gastric cancer cell lines, CDX and PDX. In addition, HLX22 in combination with HLX02 demonstrated stronger antitumor efficacy than HLX02 and HLX11 (a potential pertuzumab biosimilar) combination treatment both in vitro and in vivo. CONCLUSIONS: These results suggested that the application of non-competing antibodies HLX22 and HLX02 targeting HER2 subdomain IV together may be of substantial benefit to gastric cancer patients who currently respond suboptimal to trastuzumab therapy.

Evaluation of the PIK3 pathway in peripheral T-cell lymphoma and NK/T-cell lymphoma.

Peripheral T-cell lymphomas (PTCL) and natural killer (NK)/T-cell lymphomas (NKTCL) are a heterogeneous group of aggressive malignancies with dismal outcomes and limited treatment options. While the phosphatidylinositol 3-kinase (PIK3) pathway has been shown to be highly activated in many B-cell lymphomas, its therapeutic relevance in PTCL and NKTCL remains unclear. The aim of this study is to investigate the expression of PIK3 and phosphatase and tensin homolog (PTEN) in these subtypes of lymphoma and to identify potential therapeutic targets for clinical testing. Therefore, the expression of PIK3α, PIK3ß, PIK3γ, PIK3δ and PTEN was analyzed in 88 cases of PTCL and NKTCL samples by immunohistochemistry. All PTCL and NKTCL samples demonstrated high expression of PIK3 isoforms. In particular, high PIK3α expression was significantly associated with poor survival, even after adjustment for age, International Prognostic Index (IPI) score and anthracycline-based chemotherapy in first line. Notably, copanlisib, a pan-class I inhibitor with predominant activities towards PIK3α and PIK3δ isoforms, effectively inhibited phosphorylation of AKT, 4E-BP-1 and STAT3, causing G0 /G1 cell cycle arrest and resulting in suppression of tumour cell growth in vitro and in vivo. This study provides evidence that targeting the PIK3 pathway, particularly simultaneous inhibition of PIK3α and δ, could be a promising approach for the treatment of PTCL and NKTCL.

BAY 1125976, a selective allosteric AKT1/2 inhibitor, exhibits high efficacy on AKT signaling-dependent tumor growth in mouse models.

The PI3K-AKT-mTOR signaling cascade is activated in the majority of human cancers, and its activation also plays a key role in resistance to chemo and targeted therapeutics. In particular, in both breast and prostate cancer, increased AKT pathway activity is associated with cancer progression, treatment resistance and poor disease outcome. Here, we evaluated the activity of a novel allosteric AKT1/2 inhibitor, BAY 1125976, in biochemical, cellular mechanistic, functional and in vivo efficacy studies in a variety of tumor models. In in vitro kinase activity assays, BAY 1125976 potently and selectively inhibited the activity of full-length AKT1 and AKT2 by binding into an allosteric binding pocket formed by kinase and PH domain. In accordance with this proposed allosteric binding mode, BAY 1125976 bound to inactive AKT1 and inhibited T308 phosphorylation by PDK1, while the activity of truncated AKT proteins lacking the pleckstrin homology domain was not inhibited. In vitro, BAY 1125976 inhibited cell proliferation in a broad panel of human cancer cell lines. Particularly high activity was observed in breast and prostate cancer cell lines expressing estrogen or androgen receptors. Furthermore, BAY 1125976 exhibited strong in vivo efficacy in both cell line and patient-derived xenograft models such as the KPL4 breast cancer model (PIK3CAH1074R mutant), the MCF7 and HBCx-2 breast cancer models and the AKTE17K mutant driven prostate cancer (LAPC-4) and anal cancer (AXF 984) models. These findings indicate that BAY 1125976 is a potent and highly selective allosteric AKT1/2 inhibitor that targets tumors displaying PI3K/AKT/mTOR pathway activation, providing opportunities for the clinical development of new, effective treatments.

Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

Pan-PI3K inhibition with copanlisib overcomes Treg- and M2-TAM-mediated immune suppression and promotes anti-tumor immune responses.

The PI3K pathway is one of the most frequently altered signaling pathways in human cancer. In addition to its function in cancer cells, PI3K plays a complex role in modulating anti-tumor immune responses upon immune checkpoint inhibition (ICI). Here, we evaluated the effects of the pan-Class I PI3K inhibitor copanlisib on different immune cell types in vitro and on tumor growth and immune cell infiltration in syngeneic murine cancer models. Intermittent treatment with copanlisib resulted in a strong in vivo anti-tumor efficacy, increased tumor infiltration of activated T cells and macrophages, and increased CD8+ T cell/regulatory T cell and M1/M2 macrophage ratios. The strong in vivo efficacy was at least partially due to immunomodulatory activity of copanlisib, as in vitro these murine cancer cells were resistant to PI3K inhibition. Furthermore, the combination of copanlisib with the ICI antibody anti-PD-1 demonstrated enhanced anti-tumor efficacy in both ICI-sensitive and insensitive syngeneic mouse tumor models. Importantly, in an ICI-sensitive model, combination therapy resulted in complete remission and prevention of tumor recurrence. Thus, the combination of ICIs with PI3K inhibition by intermittently dosed copanlisib represents a promising new strategy to increase sensitivity to ICI therapies and to treat human solid cancers.

Doxorubicin-induced activation of NF-κB in melanoma cells is abrogated by inhibition of IKKß, but not by a novel IKKα inhibitor.

Drug resistance is arguably the most important challenge in cancer therapy. Here, doxorubicin induced profound of NF-κB activation in melanoma cells with a maximum (3.5-fold) at concentrations relevant in vivo. This was followed by transcriptional induction of several gene products involved in tumor progression. A novel IKKα inhibitor (BAY32-5915) was identified and characterized, and doxorubicin-induced NF-κB activation was assessed following inhibition of IKKα or IKKß by small-molecular compounds. While the IKKα inhibitor did not affect doxorubicin-induced NF-κB activation, this process was completely abrogated when the IKKß inhibitor, KINK-1, was used. Moreover, inhibition of IKKß, but not IKKα, led to significantly increased apoptosis in response to doxorubicin. Our results indicate that the net outcome of chemotherapy is difficult to predict and may even involve mechanisms conferring chemoresistance. In case of doxorubicin-induced NF-κB activation, blocking IKKß, but not IKKα, by small molecules can antagonize this activity and, thus, increase chemosensitivity.

Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/ß/δ treatment.

Combining immune checkpoint therapy (ICT) and targeted therapy holds great promises for broad and long-lasting anti-cancer therapies. However, combining ICT with anti-PI3K inhibitors have been challenging because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we find that intermittent but not daily dosing of a PI3Kα/ß/δ inhibitor, BAY1082439, on Pten-null prostate cancer models could overcome ICT resistance and unleash CD8+ T cell-dependent anti-tumor immunity in vivo. Mechanistically, BAY1082439 converts cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFNα/IFNγ pathway activation, ß2-microglubin expression and CXCL10/CCL5 secretion. With its preferential regulatory T cell inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8+ T cells, most likely via tertiary lymphoid structures. Once primed, tumors remain T cell-inflamed, become responsive to anti-PD-1 therapy and have durable therapeutic effect. Our data suggest that intermittent PI3K inhibition can alleviate Pten-null cancer cell-intrinsic immunosuppressive activity and turn "cold" tumors into T cell-inflamed ones, paving the way for successful ICT.

Copanlisib synergizes with conventional and targeted agents including venetoclax in B- and T-cell lymphoma models.

Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Kα and PI3Kδ. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18 phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas.

Cotargeting the Cell-Intrinsic and Microenvironment Pathways of Prostate Cancer by PI3Kα/ß/δ Inhibitor BAY1082439.

Targeting the PI3K pathway is a promising strategy for treating prostate cancers with PTEN-loss. However, current anti-PI3K therapies fail to show long lasting in vivo effects. We find that not only the PI3Kα- and PI3kß-isoforms, but also PI3Kδ, are associated with the epithelial-mesenchymal transition (EMT), a critical process distinguishing indolent from aggressive prostate cancer. This suggests that cotargeting PI3Kα/ß/δ could preempt the rebound activation of the parallel pathways induced by α- or ß-isoform-selective inhibitor and prevent EMT. Indeed, BAY1082439, a new selective PI3Kα/ß/δ inhibitor, is highly effective in vivo in inhibiting Pten-null prostate cancer growth and preventing EMT in the mutant Pten/Kras metastatic model. The anti-PI3Kδ property of BAY1082439 further blocks B-cell infiltration and lymphotoxin release, which are tumor microenvironment factors that promote castration-resistant growth. Together, our data suggest a new approach for the treatment of prostate cancer by targeting both tumor cells and tumor microenvironment with PI3Kα/ß/δ inhibitor. Mol Cancer Ther; 17(10); 2091-9. ©2018 AACR.

T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1.

Leukemia stem cells (LSCs) are regarded as the origins and key therapeutic targets of leukemia, but limited knowledge is available on the key determinants of LSC 'stemness'. Using single-cell RNA-seq analysis, we identify a master regulator, SPI1, the LSC-specific expression of which determines the molecular signature and activity of LSCs in the murine Pten-null T-ALL model. Although initiated by PTEN-controlled ß-catenin activation, Spi1 expression and LSC 'stemness' are maintained by a ß-catenin-SPI1-HAVCR2 regulatory circuit independent of the leukemogenic driver mutation. Perturbing any component of this circuit either genetically or pharmacologically can prevent LSC formation or eliminate existing LSCs. LSCs lose their 'stemness' when Spi1 expression is silenced by DNA methylation, but Spi1 expression can be reactivated by 5-AZ treatment. Importantly, similar regulatory mechanisms may be also present in human T-ALL.

Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth.

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.

Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression by Blocking BCR-Dependent and -Independent Activation of NF-κB and AKT.

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 CD79mut, CARD11mut, TNFAIP3mut, or MYD88mut. Inhibition of PI3Kα/δ resulted in tumor regression in an ibrutinib-resistant CD79BWT/MYD88mut patient-derived ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79Bmut-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 CD79Bmut/MYD88mut ABC-DLBCL models.

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT.

Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.

Discovery and SAR of Novel 2,3-Dihydroimidazo[1,2-c]quinazoline PI3K Inhibitors: Identification of Copanlisib (BAY 80-6946).

The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in many disease states, including tumor cells, either by growth factor receptor tyrosine kinases or by the genetic mutation and amplification of key pathway components. A variety of PI3K isoforms play differential roles in cancers. As such, the development of PI3K inhibitors from novel compound classes should lead to differential pharmacological and pharmacokinetic profiles and allow exploration in various indications, combinations, and dosing regimens. A screening effort aimed at the identification of PI3Kγ inhibitors for the treatment of inflammatory diseases led to the discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subsequent lead optimization program targeting cancer therapy focused on inhibition of PI3Kα and PI3Kß. Herein, initial structure-activity relationship findings for this class and the optimization that led to the identification of copanlisib (BAY 80-6946) as a clinical candidate for the treatment of solid and hematological tumors are described.

Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling.

The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF-MEK-ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2(+) cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.

BAY 80-6946 is a highly selective intravenous PI3K inhibitor with potent p110α and p110δ activities in tumor cell lines and xenograft models.

Because of the complexity derived from the existence of various phosphoinositide 3-kinase (PI3K) isoforms and their differential roles in cancers, development of PI3K inhibitors with differential pharmacologic and pharmacokinetic profiles would allow best exploration in different indications, combinations, and dosing regimens. Here, we report BAY 80-6946, a highly selective and potent pan-class I PI3K inhibitor with sub-nanomolar IC50s against PI3Kα and PI3Kδ. BAY 80-6946 exhibited preferential inhibition (about 10-fold) of AKT phosphorylation by PI3Kα compared with PI3Kß in cells. BAY 80-6946 showed superior antitumor activity (>40-fold) in PIK3CA mutant and/or HER2 overexpression as compared with HER2-negative and wild-type PIK3CA breast cancer cell lines. In addition, BAY 80-6946 revealed potent activity to induce apoptosis in a subset of tumor cells with aberrant activation of PI3K as a single agent. In vivo, single intravenous administration of BAY 80-6946 exhibited higher exposure and prolonged inhibition of pAKT levels in tumors versus plasma. BAY 80-6946 is efficacious in tumors with activated PI3K when dosed either continuously or intermittently. Thus, BAY 80-6946 induced 100% complete tumor regression when dosed as a single agent every second day in rats bearing HER2-amplified and PIK3CA-mutated KPL4 breast tumors. In combination with paclitaxel, weekly dosing of BAY 80-6946 is sufficient to reach sustained response in all animals bearing patient-derived non-small cell lung cancer xenografts, despite a short plasma elimination half-life (1 hour) in mice. Thus, BAY 80-6946 is a promising agent with differential pharmacologic and pharmacokinetic properties for the treatment of PI3K-dependent human tumors.

CpG directly induces T-bet expression and inhibits IgG1 and IgE switching in B cells.

CpG DNA has immunomodulatory effects, such as the suppression of allergic responses mediated by type II T cell help (T(H)2). Here we report that CpG, but not lipopolysaccharide (LPS), rapidly induces expression of T-bet mRNA in purified B cells. Up-regulation of T-bet by CpG is abrogated in mice deficient in Toll-like receptor 9 (TLR9) and MyD88, but remains intact in B cells deficient in STAT1 (signal transducer and activator of transcription 1). Interleukin 12 (IL-12) alone does not up-regulate T-bet mRNA, but greatly enhances CpG-induced T-bet expression. Furthermore, CpG inhibits immunoglobulin G1 (IgG1) and IgE switching induced by IL-4 and CD40 signaling in purified B cells, and this effect correlates with up-regulation of T-bet. Thus, CpG triggers anti-allergic immune responses by directly regulating T-bet expression via a signaling pathway in B cells that is dependent upon TLR9, independent of interferon-gamma (IFN-gamma)-STAT1 and synergistic with IL-12.