Current status of phosphoinotiside-3 kinase inhibitors in blood cancers.

PURPOSE OF REVIEW: Treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) underwent paradigm shifts, with targeted agents rapidly displacing chemotherapy. Phosphoinotiside-3 kinase (PI3K) is essential for survival and proliferation of neoplastic B cells and has proven a tractable target in NHL, with four agents receiving FDA approval in the last decade. This review summarizes key data and challenges associated with use of PI3K inhibitors in routine practice. RECENT FINDINGS: Idelalisib and duvelisib are active in CLL and indolent NHL, including in patients with high-risk features. Despite differential targeting of PI3K isoforms, they exhibit comparable efficacy and adverse event profile including autoimmune events (transaminitis, colitis, pneumonitis), mediated by Treg/Th17 imbalance. Although copanlisib, a pan-PI3K inhibitor, is associated with a distinct safety profile (hyperglycemia, hypertension), preclinical studies indicate that umbralisib, a dual inhibitor of PI3Kδ and casein kinase 1ε, may have less effect on Tregs. However, both drugs may still cause immune-mediated toxicities. SUMMARY: With close monitoring and management of adverse events, PI3K inhibitors continue to have a role in therapy of R/R CLL and NHL. Strategies to mitigate adverse events and increase efficacy of PI3K inhibitors include time-limited combination approaches, intermittent dosing schedules.

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma.

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.

Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.

Pharmacologic Targeting of Mcl-1 Induces Mitochondrial Dysfunction and Apoptosis in B-Cell Lymphoma Cells in a TP53- and BAX-Dependent Manner.

PURPOSE: Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. EXPERIMENTAL DESIGN: We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. RESULTS: Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. CONCLUSIONS: Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells.

Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.

SYK inhibition thwarts the BAFF - B-cell receptor crosstalk and thereby antagonizes Mcl-1 in chronic lymphocytic leukemia.

Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

Pevonedistat, a Nedd8-activating enzyme inhibitor, sensitizes neoplastic B-cells to death receptor-mediated apoptosis.

While death receptor ligands (Fas and TRAIL) kill chemoresistant tumor cell lines, related therapies have limited clinical efficacy as single agents. Death receptor signaling is modulated by nuclear factor-κB (NFκB), a family of transcription factors which are constitutively active in B-cell malignancies. We and others have shown that pevonedistat, an investigational inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in B-cell neoplasia. Here we demonstrate that diffuse large B-cell lymphoma, particularly activated B-cell type, and primary chronic lymphocytic leukemia cells are re-sensitized to extrinsic apoptosis by pevonedistat. Pevonedistat enhanced caspase-8 processing following death receptor ligation, and downmodulated cFLIP, a NFκB-regulated protease-deficient caspase homolog. However, treatment with pevonedistat did not modulate death-inducing signaling complex in neoplastic B-cells, suggesting that they were sensitized to death ligands through the mitochondrial pathway. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies.

Targeting neddylation effectively antagonizes nuclear factor-κB in chronic lymphocytic leukemia B-cells.

Chronic lymphocytic leukemia (CLL) B-cells demonstrate both constitutive and stroma-mediated activation of nuclear factor-κB (NF-κB). NEDD8, a ubiquitin-like protein, regulates activity of Cullin-RING ubiquitin ligases (CRLs) and thus indirectly controls NF-κB activity. Inhibition of CRLs with MLN4924, an investigational agent that targets the NEDD8-activating enzyme, induces accumulation of CRL substrates, including inhibitor of NF-κB (IκB), a negative pathway modulator. We demonstrate that both continuous and pulse treatments with MLN4924 abrogate NF-κB activity in CLL B-cells ex vivo in a CD40L-expressing stromal co-culture system and identify pathways potentially responsible for resistance to MLN4924. To achieve long-lasting therapeutic effects in CLL, combination strategies are likely necessary.

The Nedd8-activating enzyme inhibitor MLN4924 thwarts microenvironment-driven NF-κB activation and induces apoptosis in chronic lymphocytic leukemia B cells.

BACKGROUND: Stromal-mediated signaling enhances NF-κB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IκBα may partially account for constitutive activation of NF-κB. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates. EXPERIMENTAL DESIGN: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-κB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry. RESULTS: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-κB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IκBα, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-κB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)-associated kinases. CONCLUSIONS: MLN4924 disrupts NF-κB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

Pro-apoptotic TP53 homolog TAp63 is repressed via epigenetic silencing and B-cell receptor signalling in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is an accumulative disorder marked by deficient apoptosis. The TP53 homolog TAp63 promotes apoptosis and chemosensitivity in solid tumours and its deregulation may contribute to CLL cell survival. We found that TAp63α was the most prevalent TP63 isoform in CLL. Compared to healthy B cells, TAp63 mRNA was repressed in 55·7% of CLL samples. TP63 promoter methylation was high in CLL and inversely correlated with TP63 protein expression in B-cell lymphoma cell lines. siRNA-mediated knockdown of TP63 resulted in partial protection from spontaneous apoptosis accompanied by reductions in PMAIP1 (NOXA), BBC3 (PUMA), and BAX mRNA in CLL cells and increased proliferation of Raji lymphoma cells. TAp63 mRNA levels were higher in CLL with unmutated IGHV. B-cell receptor (BCR) engagement led to repression of TP63 mRNA expression in malignant B cells, while pharmacological inhibition of BCR signalling prevented TP63 downregulation. MIR21, known to target TAp63, correlated inversely with TAp63 expression in CLL, and BCR-mediated downregulation of TP63 was accompanied by MIR21 upregulation in most CLL samples. Our data illustrate the pro-apoptotic function of TP63, provide insights into the mechanisms of BCR-targeting agents, and establish a rationale for designing novel approaches to induce TP63 in CLL and B-cell lymphoma.

FASN and CD36 predict survival in rituximab-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma is the most common lymphoid malignancy, as it accounts for approximately one third of all patient cases of non-Hodgkin's lymphoma. Patients with diffuse large B-cell lymphoma have markedly different treatment outcomes, suggesting a need for reliable prognostic factors and novel therapeutic approaches. De novo fatty acid synthesis is an important metabolic driver of tumor in multiple malignancies. In this retrospective study, we analyzed expression of fatty acid synthase (a key enzyme in de novo fatty acid synthesis), Spot 14 (thyroid hormone responsive Spot 14, a nuclear protein that promotes expression of genes involved in fatty acid synthesis), and CD36 (the cell surface channel for exogenous fatty acid uptake) in patients with diffuse large B-cell lymphoma and their clinical significance. We observed that overexpression of fatty acid synthase is negatively associated with overall survival (p=0.001) and progression-free period (p=0.004) in patients with diffuse large B-cell lymphoma. Multivariate analysis showed that fatty acid synthase overexpression is an independent prognostic marker of aggressive clinical course. For the first time, we report CD36 as an independent protective factor in patients treated with rituximab. Thus, fatty acid synthase and CD36 expression may serve as prognostic markers to predict response to treatment and survival in diffuse large B-cell lymphoma patients. Fatty acid synthase may also be a potential therapeutic target in lymphoid malignancies.

Chromosomal instability substantiates poor prognosis in patients with diffuse large B-cell lymphoma.

PURPOSE: The specific role of chromosomal instability (CIN) in tumorigenesis has been a matter of conjecture. In part, this is due to the challenge of directly observing chromosome mis-segregation events as well as the inability to distinguish the role of CIN, which consists of increased rates of chromosome mis-segregation, from that of aneuploidy, which is a state of nondiploid chromosome number. EXPERIMENTAL DESIGN: Here, we examine the contribution of CIN to the prognosis of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) by directly surveying tumor cells, fixed while undergoing anaphase, for evidence of chromosome mis-segregation. Hematoxylin and eosin-stained samples from a cohort of 54 patients were used to examine the relationship between frequencies of chromosome mis-segregation and patient prognosis, overall survival, and response to treatment. RESULTS: We show that a two-fold increase in the frequency of chromosome mis-segregation led to a 24% decrease in overall survival and 48% decrease in relapse-free survival after treatment. The HR of death in patients with increased chromosome mis-segregation was 2.31 and these patients were more likely to present with higher tumor stage, exhibit tumor bone marrow involvement, and receive a higher International Prognostic Index score. CONCLUSIONS: Increased rates of chromosome mis-segregation in DLBCL substantiate inferior outcome and poor prognosis. This is likely due to increased heterogeneity of tumor cells leading to a larger predilection for adaptation in response to external pressures such as metastasis and drug treatments. We propose that targeting CIN would yield superior prognosis and improved response to chemotherapeutic drugs.

Dipeptidyl peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia.

OBJECTIVE: Dipeptidyl peptidase 2 (DPP2/DPP7) is a regulator of quiescence as inhibition of DPP2 results in apoptosis of resting, but not activated lymphocytes. The purpose of the present study was to investigate the prognostic value of DPP2 inhibition and the role of DPP2 in cell cycle in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: We screened 152 peripheral blood samples from patients with CLL in an apoptosis assay with AX8819, a DPP2-specific inhibitor. The apoptotic response was correlated with B-cell receptor signaling and cell cycle and molecular prognostic factors. RESULTS: We categorized CLL into two prognostic subgroups. Inhibition of DPP2 induced apoptosis in 60% of CLL, while 40% were resistant to apoptosis. Resistance to apoptosis correlated with unmutated IgV(H) and increased ZAP-70 expression and was associated with unfavorable clinical outcomes. Sensitive CLL B cells expressed high p27, low c-Myc protein levels and decreased Syk phosphorylation, indicative of a resting phenotype. DPP2 inhibition in those cells resulted in apoptosis accompanied by enhanced phosphorylation of Syk, degradation of p27 and p130, and upregulation of c-Myc, indicative of activation and inappropriate cell cycle entry. Resistant CLL demonstrated baseline low p27 and high c-Myc protein levels and increased pSyk, indicative of an activated phenotype. Inhibition of heat shock protein 90 in this subset of CLL partially reversed apoptosis resistance. CONCLUSIONS: The DPP2 apoptosis assay provides a reliable prognostic factor in CLL. CLL B cells sensitive to DPP2 inhibition are in true G(0), while resistant CLL B-cells are partially activated. DPP2 inhibition alone or with concomitant inhibition of heat shock protein 90 warrants investigation as a therapeutic modality in CLL.

Molecular pathogenesis of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is unique among malignancies since it represents an accumulation of B-lymphocytes resistant to apoptosis. Several factors are thought to confer this unusual feature to a CLL B-cell. Misbalance between cytoplasmic pro-survival and pro-death molecules, such as Bcl-2, Mcl-1 and alike, appears to be one of the key factors defining B-cell longevity. Autocrine pathways, such as vascular endothelial growth factor-receptor pathway, also contribute to survival. The role of B-cell receptor (BCR) is less straightforward. In the last decade it became clear that CLL does not constitute a uniform disease, but, based on the prevalence of mutations in the BCR heavy chain (IgVH), can be classified into two distinct subgroups. Several molecular markers correlate with IgVH mutations. Some of them, like zeta-chain associated protein kinase, are also involved in BCR signaling and influence cell cycle. Yet the primary pathogenic event leading to increased proliferation and survival in CLL is difficult to ascertain. Molecules involved in BCR signaling pathways and cytoplasmic pro-survival players probably act in concert to confer resistance to apoptosis. In this respect, the role of the B-CLL environment, which includes nurse-like cells and T-cells, cannot be underestimated. Nurse-like cells provide stimuli necessary for perpetuation of life in CLL. On the other hand, abnormal T-cell function, whether it is excessive immunosuppression delivered by regulatory T-cells or insufficient anti-tumor immunity rendered by T-helpers, allows malignant CLL cells to go unnoticed by the cellular immune system.