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Investigating the role of Class Ia phosphoinositide-3 kinase isoforms in Mantle Cell Lymphoma.
Abstract and Figures
Mantle Cell Lymphoma (MCL) is a rare but aggressive Non-Hodgkin Lymphoma (NHL). Although
t(11;14) is a hallmark of MCL, it is insufficient for lymphomagenesis. The phosphoinositide-3 kinase
(PI3K) pathway is thought to play an important role in MCL pathogenesis and the PI3K p110δ isoform
is enriched in leucocytes making it an attractive target. Early phase trials evaluating the p110δ
selective inhibitor GS-1101 however demonstrate inferior responses in MCL compared to chronic
lymphocytic leukaemia and indolent NHL.
The relative contribution of the class Ia PI3K isoforms p110α (PIK3CA), p110β (PIK3CB) and p110δ
(PIK3CD) was therefore evaluated in MCL. Immunohistochemistry on MCL tissue microarrays
revealed that while p110δ was highly expressed, p110α showed wide variation and p110β
expression was the weakest. A significant increase in p110α expression was found with disease
progression. Although GS-1101 was sufficient to abolish B-cell receptor mediated PI3K activation,
additional p110α inhibition was necessary to abolish constitutive PI3K activation in MCL exhibiting
high p110α expression. Compared to GS-1101, GDC-0941 (p110α and p110δ inhibitor) had greater in
vitro toxicity and a high PIK3CA/PIK3CD mRNA ratio (> twice ratio in healthy B-cells) was able to
identify primary MCL samples that were resistant to GS-1101 but significantly more sensitive to
GDC—0941. This ratio also increased with disease progression. No PIK3CA or PIK3R1 activating
mutations were found. In summary, blockade of both p110α and p110δ appears to be necessary for
effective PI3K inhibition in MCL, particularly with relapse. The PIK3CA/PIK3CD mRNA ratio may help
identify those patients that are most likely to respond.
Finally, a disseminated xenograft model of human primary MCL was established in NSG mice.
Engraftment of primary MCL was demonstrated by peripheral blood flow cytometry, tissue
immunohistochemistry and FISH for t(11;14). This model is potentially valuable for pre-clinical in vivo
testing of novel drugs for this incurable disease.
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Background: The PI3K-PTEN-AKT signaling pathway is deregulated in a wide variety of cancers. GDC-0941 is a potent and selective oral inhibitor of the class I PI3K with 3 nM IC50 for the p110-alpha subunit in vitro and 28 nM IC50 in a cell-based pAKT assay and demonstrates broad activity in breast, ovarian, lung, and prostate cancer models. Methods: A Phase I dose escalation study using a 3+3 design was initiated in patients (pts) with solid tumors. GDC-0941 was given on d1, followed by 1 wk washout to study single-dose PK and PD markers. GDC-0941 was then administered qd on a 3 wk on, 1 wk off, schedule. Steady-state PK and PD were evaluated after 1 wk of continuous dosing. A separate concurrent dose-escalation arm with bid dosing was initiated after the third qd cohort. Results: Nineteen pts have been enrolled in 5 successive dose-escalation cohorts in the qd arm with dose levels up to 80 mg daily. Seven pts were enrolled in 2 cohorts in the bid arm at total daily doses of 60 and 80 mg. The most frequently reported drug-related AEs were Grade 1/2 nausea, fatigue, diarrhea, peripheral edema, and dysgeusia; no drug related grade >3 events have been reported. PK data suggest dose-proportional increases in Cmax and AUC. Potential signs of anti-tumor activity have been observed with a soft tissue sarcoma pt on-study for >176 days with stable disease (30 mg qd), an ovarian cancer pt with an on-study 2.8-fold decrease in CA-125 response to normal levels (30 mg bid) and a pt with endometrial cancer with a decrease in tumor FDG-PET uptake (80 mg qd). Conclusions: GDC-0941 is generally well-tolerated with potential signs of anti-tumor activity. Preliminary PK data suggest dose-proportional increases in exposure over the dose levels evaluated. Dose-escalation on both the qd and bid schedules continues with updated data to be presented.
[Table: see text]
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Poster Board III-880
Background
MCL is a clinically aggressive lymphoma characterized by frequent relapses and short survival. Clinical behavior and response to therapy vary considerably and are often unpredictable based on clinical factors. Prior gene and protein expression studies have demonstrated proliferation and TP53 expression as biological mechanisms that strongly influence survival (Rosenwald Cancer Cell 2003 & Katzenberger Blood 2006 and Louie Blood 1995). Recent studies have highlighted the role of the microenvironment in follicular lymphoma (FL) including numbers of lymphoma-associated-macrophages (LAM), as important prognostic factors (Dave NEJM 2004 & Farinha Blood 2005). Within most tumors, macrophages display an M2 phenotype that promotes tumor growth and angiogenesis. In MCL, the clinical impact of LAM is unknown. Our aim was to assess the prognostic impact of LAM in MCL patients from a single institution experience using tissue microarrays (TMA), immunohistochemistry and clinical correlates.
Methods
A TMA block was built with duplicate 0.6mm cores of paraffin-embedded formalin-fixed diagnostic biopsies from 185 patients treated at the BC Cancer Agency (1983-2004). A subset of cases was also analyzed for the presence of the t(11;14) by FISH. Primary and subsequent therapy included multiple regimens: single or multi-agent chemotherapy with or without rituximab, stem cell transplant, radiation and therapeutic splenectomy. Standard immunohistochemistry was performed with CD20, CCND1, CD68, CD163 (an M2 marker), CD34, TP53 and Ki67. TP53 and Ki67 IHC were scored using image analysis (VIAS). Univariate and multivariate correlates of overall survival (OS) were determined using SPSS software.
Results
The median follow-up of living patients was 5.75 years. The IPI predicted OS (p=0.0013). Numbers of cases with interpretable staining varied for the different biomarkers (range 157 to 180). All 185 cases were either CCND1 positive and/or t(11;14) FISH positive. Both proliferation (quartiles of Ki67+ cells) and TP53 (dichotomized based on cut-off >40%) were significant predictors of OS independent of IPI (OS, p=0.0002). Cases showing more than 5% infiltrating CD68+ cells (18/185) had a 4-y OS of 5% vs. 35% (p=0.0081). These CD68+ cases significantly correlated with 12 cases showing more than 5% infiltrating CD163+ cells (x2=0.001), which also significantly influenced OS (p=0.008). 17 cases had increased microvessel density (MVD), defined by CD34+ cells, which correlated with inferior OS (p=0.02). Interestingly, cases with increased MVD often had >5% CD163+ cells (x2=0.05), but not increased CD68+ cells. Multivariate analysis including IPI, TP53, Ki67, CD68, CD163 and CD34, showed IPI (RR=1.8, 95%CI=1.0-3.1, p=0.047), TP53 (RR=2.3, 95%CI=1.3-4.3, p=0.006), Ki67 (RR=2.2, 95%CI=1.2-4.2, p=0.012) and CD68 (RR=2.1, 95%CI=1.1-3.8, p=0.022) to be independent predictors of OS. Finally, in the 27 patients who received rituximab at some time during their disease course, increased CD68+ cells did not significantly affect OS (p=0.1), suggesting that, similar to FL, the prognostic effect of LAM may be abolished by rituximab.
Conclusions
The number of LAMs is an important prognostic factor in MCL, independent of clinical parameters, proliferation and TP53 expression. Expression of CD163, described as an M2 polarized macrophages, also predicts survival and correlates with increased angiogenesis. These preliminary data suggest that the use of rituximab may abrogate the prognostic impact of LAM in MCL. In summary, similar to other lymphoma subtypes, the microenvironment is important in MCL biology and prognosis and may be an important new target for therapy in this aggressive disease.
Disclosures
Connors: Roche Canada (F Hoffmann-La Roche): Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Mantle cell lymphoma (MCL) is a type of aggressive B-cell non-Hodgkin lymphoma characterized by frequent resistance to conventional chemotherapy. Currently, there is no standard of care for the treatment of MCL, and patient prognosis is poor. It is well known that the tumor microenvironment plays an important role in tumor cell growth and resistance to chemotherapy. However, little is known about the microenvironment and its influence in MCL. In this study we investigated the role of IL-6 and its signaling in the growth, survival, and development of drug resistance in MCL, as IL-6 is an important cytokine for B cells and multiple myeloma. We found that the membrane IL-6 receptor gp130 is generally expressed in established MCL cell lines and in primary lymphoma cells from patients, which can be upregulated by stress such as serum starvation or low-dose chemotherapy drug treatment. Some but all not MCL cells also secrete IL-6 and/or IL-6 soluble receptor gp80. Although IL-6 and its signaling pathway do not affect MCL growth in vitro, they play an important role in MCL survival and resistance to chemotherapy drugs. Neutralizing IL-6 and/or blocking IL-6 receptors in IL-6- or gp80-secreting MCL cells increased their sensitivity to chemotherapy drug- and serum starvation-induced apoptosis. For MCL cells that do not secrete IL-6 or gp80, low doses of exogenous IL-6 or gp80 protected them from chemotherapy drug-induced apoptosis. Because T cells, macrophages, and bone marrow stromal cells (BMSCs) secrete IL-6 and/or gp80, coculture of MCL cells with peripheral blood mononuclear cells or BMSCs protected MCL cells from chemotherapy drug-induced growth inhibition and apoptosis, which can be abrogated by anti-IL-6, anti-gp80, and/or anti-gp130 antibodies. Knocking down gp80 in gp80high MCL cells rendered the cells more sensitive to chemotherapy drug-induced apoptosis, even in the presence of exogenous IL-6. Overexpression of gp80 in gp80low IL-6+ MCL cells provided protection of MCL cells from chemotherapy drug-induced apoptosis. Next, Jak2 or STAT3 inhibitors completely abrogated IL-6-mediated protection of MCL cells from apoptosis, whereas PI3K or MEK inhibitors partially abrogated IL-6-mediated protection of MCL cells from apoptosis. Furthermore, gp80-overexpressing, gp80low IL-6+ MCL cells grew faster than vector control or parental cells in the SCID mouse model, and immunohistochemistry staining showed strong surface gp80 and nuclear phosphorylated STAT3 in gp80-overexpressing MCL tumor cells. Thus, these results clearly show that IL-6 and gp80, derived from MCL cells themselves or from cells in the microenvironment, play a pivotal role in MCL cell survival and drug resistance. Although IL-6 activates Jak2/STAT3, PI3K/AKT, and MEK/Erk signaling pathways, STAT3 signaling may play an important role in mediating IL-6-induced protection of MCL against chemotherapy drug-induced apoptosis. This study suggests that targeting IL-6 and its signaling pathway may improve the efficacy of chemotherapy in MCL patients.
Disclosures
Wang: Celgene: Honoraria, Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding.
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The clinical efficacy of mTOR inhibition in MCL is limited by known resistance pathways mediated through IRS-1 and mTORC2. Simultaneous inhibition of other molecules downstream of the B cell receptor, such as PI3Kδ, may abrogate such negative feedback mechanisms. PI3Kδ inhibition using GS-1101 has demonstrated early efficacy in MCL. Taken together, the combination of mTORC1 and PI3Kδ inhibition may represent a rationale combination to test in MCL.
To this end, we utilized a panel of B cell lymphoma lines including established MCL cell lines (Granta, Jeko, Mino, Rec-1, HBL-2, Z-138), cytarabine resistant MCL lines (MinoAraCR, JekoAraCR, Rec-1AraCR, HBL-2AraCR) and primary MCL cells isolated from patients. In all cell lines, dose-finding experiments using GS-1101 and the mTOR inhibitors temsirolimus and everolimus were performed in triplicates. Cell viability was determined using an Alamar Blue reduction assay. Proteins downstream of PI3K – mTOR signaling were evaluated by western blot analysis. Synergy between the agents was evaluated using Laska et al's model–free test. For in vivo studies, severe combined immunodeficiency mice were injected with 10×106 Z-138 cells on day 0. GS-9820, a PI3Kδ inhibitor optimized for murine studies, was used in lieu of GS-1101. Upon detection of tumor engraftment, animals were divided into 6 groups, each containing 5 mice; Control, GS-9820 at 10 and 20mg/kg/dose, temsirolimus at 10 and 20mg/kg/dose, and GS-9820 plus temsirolimus at 10mg/kg/dose each. GS-9820 was administered by gastric lavage twice daily on days +15 to +19 and +22 to +26. Temsirolimus was administered via tail vein injection on days +15, +17, +19, +22, +24, and +26. Tumor measurements were used to determine therapeutic activity.
The initial screen of lymphoma histologic subtypes demonstrated that cell viability was reduced across Burkitt, diffuse large B cell and MCL lines exposed to GS-1101. In MCL lines, the cell viabilities observed after 48 h treatments with GS-1101 (5uM) were 80% ± 6.9, 66% ± 2.2 and 68% ± 4.7 in Granta, Jeko and Rec-1 cells respectively. No difference was observed in cytarabine resistant cells suggesting non-cross resistance with cytarabine. The activity in primary MCL cells was similar using GS-1101 (5uM) [viability range 55%-65%] while peripheral blood mononuclear cells (PBMCs) appeared less sensitive to GS-1101 [78% ± 2.4].
Both mTOR inhibitors provided moderate reductions in viability after 48 h exposures. Compared to untreated controls, the viabilities of Granta, Jeko and Rec-1 cell lines after 48 h exposures to temsirolimus (5nM) were 73% ± 1.3, 53% % ± 6.9 and 54% ± 2.0 respectively as well as 68% ± 2.9, 50% ± 7.4 and 55% ± 2.0 respectively after everolimus (5nM). Similar results were observed in primary MCL cells using temsirolimus (5nM) [range 80%-85%] while PBMCs were largely unaffected [90% ± 2.2].
The combination of GS-1101 and either mTOR inhibitor produced largely additive reductions in cell viability. Synergistic interactions were observed in Rec-1 cells for 8 dose combinations of GS-1101 (0.1–5.0uM) and either temsirolimus (1–5nM) or everolimus (1–5nM) (unadjusted p < 0.05 for all 8 combinations). Evidence of synergy was insufficient at any combination after adjustment for multiple comparisons over the 3 cell lines. Sequential administration using 24 h pretreatment with each agent was evaluated; no benefit over simultaneous administration was demonstrated. Consistent with known mechanisms of action, immunoblotting revealed decreased 4EBP1 and S6K phosphorylation with mTOR inhibition while PI3K inhibition consistently decreased Akt phosphorylation.
In vivo, GS-9820 appears active in the Z-138 xenografts at early time points. Tumor size was reduced to 60% ± 5.5 of control at day 18 and 23 using either 10 or 20 mg/kg of GS-9820. Testing of GS-9820 in combination with temsirolimus in this model is ongoing.
Our findings indicate that PI3Kδ inhibition using GS-1101 and GS-9820 is active in vitro and also in a MCL murine xenograft. GS-1101 in combination with mTORC1 inhibition largely produced additive in vitro anti-lymphoma effects in MCL. Ongoing work is aimed at understanding the differences in molecular events downstream of PI3K and mTOR inhibition comparing Rec-1 cells, where synergy was demonstrated, with other cell lines to provide insight into optimal therapeutic combinations and to determine in which molecularly defined subsets of MCL they may be most active.
Disclosures
Johnson: Gilead Sciences: Employment. Lannutti:Gilead Sciences Inc: Employment.
Background Despite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110δ catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy. Principal Findings We show that p110δ expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110δ gene transcription appears to be constitutive and not subject to acute stimulation. 5′RACE experiments revealed that p110δ mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110δ transcripts are higher in leukocytes compared to non-leukocytes, with the p110δ transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110δ gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a. Conclusion This study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110δ.
The activation of antigen receptors triggers two important signalling pathways originating from phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]. The first is phospholipase Cgamma (PLCgamma)-mediated hydrolysis of PtdIns(4,5)P(2), resulting in the activation of Ras, protein kinase C and Ca(2+) flux. This culminates in profound alterations in gene expression and effector-cell responses, including secretory granule exocytosis and cytokine production. By contrast, phosphoinositide 3-kinases (PI3Ks) phosphorylate PtdIns(4,5)P(2) to yield phosphatidylinositol(3,4,5)-trisphosphate, activating signalling pathways that overlap with PLCgamma or are PI3K-specific. Pathways that are PI3K-specific include Akt-mediated inactivation of Foxo transcription factors and transcription-independent regulation of glucose uptake and metabolism. The p110delta isoform of PI3K is the main source of PI3K activity following antigen recognition by B cells, T cells and mast cells. Here, we review the roles of p110delta in regulating antigen-dependent responses in these cell types.
B-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-kappaB and NFAT, are involved in regulating BLyS expression through at least one NF-kappaB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-kappaB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-kappaB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 mu/ml (3.8 mu M); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50, values in the range of 1.7-8.4 mu g/mI. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 > 30 mu g/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 mu g/ml; 1.40 mu M) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.
We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.