Abstract In previous studies we demonstrated that targeting the nuclear exporter protein exportin-1 (CRM1/XPO1) by a selective inhibitor of nuclear export (SINE) compound is a viable therapeutic ...strategy against Non-Hodgkin Lymphoma (NHL). Our studies along with pre-clinical work from others led to the evaluation of the lead SINE compound, selinexor, in a phase 1 trial in patients with CLL or NHL ( NCT02303392 ). Continuing our previous work, we studied combinations of selinexor-dexamethasone (DEX) and selinexor-everolimus (EVER) in NHL. Combination of selinexor with DEX or EVER resulted in enhanced cytotoxicity in WSU-DLCL2 and WSU-FSCCL cells which was consistent with enhanced apoptosis. Molecular analysis showed enhancement in the activation of apoptotic signaling and down-regulation of XPO1. This enhancement is consistent with the mechanism of action of these drugs in that both selinexor and DEX antagonize NF-κB (p65) and mTOR (EVER target) is an XPO1 cargo protein. SINE compounds, KPT-251 and KPT-276, showed activities similar to CHOP (cyclophosphamide–hydroxydaunorubicin–oncovin–prednisone) regimen in subcutaneous and disseminated NHL xenograft models in vivo . In both animal models the anti-lymphoma activity of selinexor is enhanced through combination with DEX or EVER. The in vivo activity of selinexor and related SINE compounds relative to ‘standard of care’ treatment is consistent with the objective responses observed in Phase I NHL patients treated with selinexor. Our pre-clinical data provide a rational basis for testing these combinations in Phase II NHL trials.
Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated ...activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.
Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth
We hypothesized that combining selinexor with ...antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity.
, selinexor increased
and
gene expression in leukocytes and induced
gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (
< 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (
≤ 0.050) and CD4
T cells with a Th1 phenotype (
≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity.
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The present study investigated the intracellular regulation of E-cadherin in ovarian carcinoma. E-cadherin expression and regulation by Snail and Pak1 were studied in ES-2 and OVCAR-3 ovarian cancer ...cells in vitro. Twist1, Zeb1 and Vimentin mRNA expression and HIF-1α protein expression were analyzed in 80 and 189 clinical specimens, respectively. OVCAR-3 cells incubated with an anti-E-cadherin antibody formed smaller and looser spheroids compared to controls. Snail silencing using Small Hairpin RNA in ES-2 cells reduced invasion and MMP-2 activity, with unaltered cellular morphology. Using dominant negative (DN) and constitutively active (CA) Pak1 constructs, we found that DN Pak1 ES-2 and OVCAR-3 clones had reduced attachment to matrix proteins, invasion and MMP-2 activity compared to CA and wild-type cells. DN Pak1 ES-2 cells also bound less to LP9 mesothelial cells. DN Pak1 OVCAR-3 cells had lower Vimentin levels. Snail expression was lower in cultured effusions compared to primary carcinomas, and was cytoplasmic rather than nuclear. Twist1 (P < 0.001), Zeb1 (P = 0.003) and Vimentin (P = 0.03) mRNA expression was significantly higher in solid metastases compared to primary carcinomas and effusions. HIF-1α protein expression was lower in effusions compared to primary carcinomas and solid metastases (P = 0.033). Our data suggest that the previously reported E-cadherin re-expression in ovarian carcinoma effusions is regulated by Pak1. The transient nature of E-cadherin expression during ovarian carcinoma progression is probably the result of partial epithelial-to-mesenchymal transition (EMT) and the reverse process of mesenchymal-to-epithelial-like transition (MET). Expression of the EMT-related molecules Twist, Zeb1, Vimentin and HIF-1α is anatomic site-dependent in ovarian carcinoma.
Unlike most epithelial cancers, E-cadherin expression is upregulated in ovarian carcinoma effusions compared with corresponding primary tumors. In the present study, we analyzed the anatomic ...site-specific expression of transcription factors that negatively regulate E-cadherin in ovarian carcinoma. Using reverse-transcription polymerase chain reaction, mRNA in situ hybridization, and Western blotting, we analyzed the expression and localization of the Snail, Slug, and SIP1 transcription factors and E-cadherin in 78 effusions, 41 primary carcinomas, and 15 solid metastases. Slug mRNA and protein expression was highest in metastases (p=0.042 and p<0.001, respectively). Snail mRNA was comparable at all anatomic sites, but higher protein expression was found in primary tumors and solid metastases compared with effusions (p<0.001). SIP1 mRNA expression was higher in effusions (p<0.001) compared to other sites. Confocal microscopy analysis of fresh and cultured cells from effusion specimens revealed cytoplasmic localization of the Snail protein in primary tumor cells, with a nuclear shift following culturing of these cells. In conclusion, E-cadherin and its negative regulators show site-dependent expression in ovarian carcinoma. In solid tumors, E-cadherin is negatively regulated by Snail and Slug. In effusions, SIP1 may be the main regulator of E-cadherin, but with a lesser level of suppression compared with primary tumors and solid metastases.
BackgroundAgonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of ...an agonistic antibody compared with natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-224, an allogeneic red cell therapeutic genetically engineered to express hundreds of thousands of copies of 4-1BBL and IL-12 in their natural conformation on the cell surface. RTX-224 is designed to activate four key target cell types: CD4+ and CD8+ T cells, antigen presenting cells and NK cells for a broad and effective anti-tumor response while providing improved safety due to the restricted biodistribution of red blood cells to the vasculature and spleen. Here we investigated the potential efficacy and mechanism of action of RTX-224 using the mouse surrogate mRBC-224.MethodsmRBC-224 was administered intravenously (i.v.) to normal or tumor-bearing mice (B16F10 tumor models). Blood, spleen and tumors were harvested and the pharmacodynamic effects of mRBC-224 on immune cells were evaluated.ResultsmRBC-224 administered to mice inoculated i.v. with B16F10 melanoma reduced the number of metastases (p<0.0001 and 76.8% tumor growth inhibition on Day 14). This was accompanied by increased proliferation (Ki67+) and cytotoxicity (GzmB+) of tumor-infiltrating CD8+ T cells and NK cells, and an increased CD8+ effector memory (TEM) phenotype. Similarly, mRBC-224 reduced tumor growth in the B16F10 s.c. model (p<0.0001 and 56.2% tumor growth inhibition on Day 9), and this was associated with increased frequency of activated (MHC-II+) tumor-infiltrating macrophages. Consistent with the known biodistribution of red cells, mRBC-224 did not distribute to the tumor but was predominantly localized in the blood and spleen raising the question about mRBC-224 mechanism of action in mediating antitumor responses. In normal and B16F10 s.c. tumor-bearing mice, mRBC-224 induced the activation of CD8+ T cells, NK cells and monocytes/macrophages in blood and spleen in a dose-dependent manner. PD studies in the tumor suggest that these activated immune cells are capable of trafficking from blood/spleen to the tumor. These results align with published data suggesting that activated T cells in the spleen or blood can replenish exhausted tumor-infiltrating cells.ConclusionsTaken together, these data unveil the mechanism of action of mRBC-224 and suggest that mRBC-224 activate immune cells in the spleen and blood, leading to their trafficking into the tumor microenvironment to promote efficacy.
Summary The AKT signaling pathway is crucial for cancer cell survival. The objective of this study was to analyze the expression and clinical role of this pathway in serous ovarian carcinoma. ...Phospho-AKT and phospho–mammalian target of rapamycin protein expression was studied in 269 ovarian carcinomas (159 effusions, 38 primary carcinomas, 72 solid metastases) using immunohistochemistry. The association between AKT, mammalian target of rapamycin, and DJ-1 in effusions was quantitatively analyzed using flow cytometry. AKT phosphorylation status in effusions was further studied using Western blotting. Phospho-AKT and phospho–mammalian target of rapamycin were detected in the majority of tumors at all anatomical sites. Phospho-AKT expression in effusions was higher in grade 3 versus grades 1 and 2 tumors ( P = .013). Flow cytometry analysis showed association between AKT, mammalian target of rapamycin, and DJ-1 expression ( P < .001). Higher phospho-AKT Thr308/pan-AKT ratio by Western blotting was associated with more advanced International Federation of Gynecology and Obstetrics stage ( P = .018) and a trend for poor response to chemotherapy at first disease recurrence ( P = .051). Higher phospho–mammalian target of rapamycin protein expression in effusions by immunohistochemistry was associated with poor progression-free survival for patients with postchemotherapy effusions ( P = .005). Phospho–mammalian target of rapamycin was an independent predictor of poor progression-free survival for patients with postchemotherapy effusions ( P = .03). The association between activated AKT and mammalian target of rapamycin expression and clinicopathologic parameters of aggressive disease, including shorter patient survival, provides further evidence regarding the central role of this signaling pathway in ovarian carcinoma.
BackgroundAgonist antibodies and recombinant cytokines have had limited success in the clinic due to three factors: severe toxicity leading to a narrow therapeutic index, the diminished activity of ...an agonistic antibody compared with natural ligand, and the lack of multiple signals needed to effectively activate most cell types. To address these limitations, Rubius Therapeutics has developed RTX-240, an allogeneic cellular therapy using red blood cells genetically engineered to express 4-1BBL and IL-15/IL-15Ra fusion (IL-15TP) in their natural conformation on the cell surface. RTX-240 is designed to recapitulate human biology by broadly stimulating adaptive and innate immunity to generate an anti-tumor response and provide improved safety due to the restricted biodistribution of red blood cells to the vasculature. Here we demonstrate that RTX-240 is highly active in preclinical models.MethodsPBMCs or NK cells were treated with RTX-240 in vitro. mRBC-240 was used for in vivo studies.ResultsTreatment of either PBMCs or isolated NK cells with RTX-240 induced a dose-dependent increase in NK cell activation, proliferation and functionality. These effects were further enhanced with increased 4-1BBL and IL-15TP expression on the surface of RTX-240. NK cell counts, NKp44 and Trail expression were increased 150, 4.6 and 6-fold over media control, respectively. Activation of NK cells with RTX-240, followed by incubation with K562 targets enhanced NK cell cytotoxicity (1.3-2.8 over control), that was accompanied by increased NK cell activation (CD69) and degranulation (CD107a) (3.1-fold and 1.9-fold, respectively). RTX-240-activated NK cells showed higher frequency of CD56dim/CD16+ NK cells, which have been reported to induce natural and ADCC-dependent cytotoxicity. Correspondingly, RTX-240 promoted enhanced ADCC-induced killing of Raji cells when combined with anti-CD20 mAb (1.4-fold over control). Intravenous administration of mRBC-240 to a B16F10 intravenous lung metastases model led to NK cell expansion on Day 4 (3.8-fold over control). These NK cells were cytotoxic (Granzyme B+) and highly proliferative (Ki67+) (1.4-fold and 18.8-fold over control, respectively). Treatment with mRBC-240 increased the frequency of terminally differentiated NK cells (NK1.1+/CD11b+/CD27-/KLRG1+) in the tumor (2.1-fold increase over control). Terminally differentiated NK cells are highly cytotoxic and their frequency in the tumor was strongly correlated with efficacy in this model (p=0.0001).ConclusionsTaken together, these data indicate that RTX-240 promotes NK cell activity and functionality in preclinical models. RTX-240 has now entered a first-in-human Phase 1 trial for the treatment of patients with relapsed/refractory or locally advanced solid tumors, with a planned arm evaluating RTX-240 in relapsed/refractory acute myeloid leukemia.
Exportin-1 (XPO1) is a nuclear export protein with >220 cargo proteins, including tumor suppressors and cell cycle modulators. Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound ...that has been administered to >900 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. Selinexor blocks nuclear export of NFAT1c, STAT1 and STAT3, which are implicated in regulating the inhibitory T cell receptor PD-1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and that combination treatment with anti-PD-1 or anti-PD-L1 would thereby enhance the ability of selinexor to elicit antitumor activity.Selinexor increased PD-1 gene expression by ~2-fold in normal lymphocytes and induced PD-L1 gene expression in tumor cell lines. Mice bearing syngeneic colon tumors (colon26) treated with selinexor and anti-PD-1 for 2 weeks demonstrated a significant reduction in tumor growth rate (P < 0.05), while monotherapy with either agent had no significant effect on tumor growth. Similar results were obtained in mice bearing syngeneic B16F10 melanoma tumors, whereby combined treatment with selinexor + anti-PD-1 was superior to either single agent alone (p < 0.034). Combined therapy of mice bearing B16F10 tumors with selinexor and anti-PD-L1 was similarly effective, with significantly smaller tumors at the study endpoint (p < 0.001). No weight loss or signs of toxicity were evident in any in vivo study.Immunophenotypic analysis by flow cytometry revealed that selinexor alone or in combination with anti-PD-1/anti-PD-L1 significantly increased the percentage of splenic NK cells (p≤0.050), while selinexor ± anti-PD-L1 significantly increased the percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p < 0.001). These data indicate that the efficacy of selinexor may be enhanced by disrupting the pre-existing PD-1/PD-L1 signaling in effector cells (T and NK cells).Altogether, these data suggest that the efficacy of selinexor in combination with anti-PD-1 or anti PD L1 in mouse syngeneic tumor models may be due to both disrupting immunosuppressive PD-1/PD-L1 signaling and increasing the frequency of potentially tumor reactive NK cells and Th1 T cells. This provides a rational basis for this treatment combination as a novel therapeutic approach for advanced cancer.
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