SummaryCereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4CRBN. T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4CRBN substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4CRBN is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4CRBN with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4CRBN, leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

• CC-122 is a novel agent for DLBCL with antitumor and immunomodulatory activity.• CC-122 binds CRBN and degrades Aiolos and Ikaros resulting in a mimicry of IFN signaling and apoptosis in DLBCL.Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros.Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-a, -b, and -g production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide. (Blood. 2015;126(6):779-789)

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

The echinocandins are a class of semisynthetic natural products that target ␤-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibited in vitro activity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GS in vitro, and there was a strong correlation between enzyme inhibition and in vitro antifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants of Saccharomyces cerevisiae with reduced susceptibility to the piperazinyl-pyridazinones had substitutions in FKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model of Candida glabrata infection.

Lenalidomide is an immunomodulatory agent that has demonstrated clinical benefit for patients with relapsed or refractory mantle cell lymphoma (MCL); however, despite this observed clinical activity, the mechanism of action (MOA) of lenalidomide has not been characterized in this setting. We investigated the MOA of lenalidomide in clinical samples from patients enrolled in the CC-5013-MCL-002 trial (NCT00875667) comparing single-agent lenalidomide versus investigator's choice single-agent therapy and validated our findings in pre-clinical models of MCL. Our results revealed a significant increase in natural killer (NK) cells relative to total lymphocytes in lenalidomide responders compared to non-responders that was associated with a trend towards prolonged progression-free survival and overall survival. Clinical response to lenalidomide was independent of baseline tumour microenvironment expression of its molecular target, cereblon, as well as genetic mutations reported to impact clinical response to the Bruton tyrosine kinase inhibitor ibrutinib. Preclinical experiments revealed lenalidomide enhanced NK cell-mediated cytotoxicity against MCL cells via increased lytic immunological synapse formation and secretion of granzyme B. In contrast, lenalidomide exhibited minimal direct cytotoxic effects against MCL cells. Taken together, these data provide the first insight into the clinical activity of lenalidomide against MCL, revealing a predominately immune-mediated MOA.

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Background: CC-122 is a first in class PPMTM pleiotropic pathway modifier compound with multiple biological activities including potent anti-proliferative activity against B lineage cells, anti-angiogenic activity and immunomodulatory effects. CC-122 binds cereblon, and promotes ubiquitination of lymphoid transcription factors Ikaros and Aiolos, leading to their subsequent degradation resulting in activation of T cells. The immunological properties of CC-122 including effects on T cell subset number in vivo and T cell cytokine production ex vivo was explored in subjects with advanced aggressive non-Hodgkin Lymphoma (NHL) and Multiple Myeloma (MM) enrolled in a Phase 1b trial (NCT01421524) at 3 mg QD and 4 and 5 mg 5/7 days dosed in 28 day cycles until disease progression. Methods: As of June 25, 2015, 76 total DLBCL and MM subjects were enrolled in the expansion phase of the study. Assessments for T cell subset numbers were performed at screening (baseline), cycle 1 day 15 (C1D15), cycle 1 day 22, cycle 2 day 15 and cycle 2 day 22 by flow cytometric immunophenotyping of fresh whole blood. Ex vivo whole blood T cell activation as measured by IL-2, IL-6, IFNg and GM-CSF cytokine production was performed using the anti-CD3 TruCulture Assay. Changes from baseline were evaluated using the t test with p<0.05 considered significant. Results: T cell subsets which were significantly changed are shown in italics in Table 1. In MM subjects (n=19-21) and DLBCL subjects (n=30-31), CC-122 treatment significantly expanded several T cell activator and memory T cell subsets while decreasing naïve T cells. A single dose of CC-122 on C1D1 activated T cells as measured in an ex vivo T cell activation assay in MM subjects (n=6-13) and DLBCL subjects (n=5-12) (Table 2). In addition, we evaluated potential correlations of clinical outcome with baseline biomarker and biomarker changes upon CC-122 treatment. Table 1. MM n=19-21 NHL n=30-31 T cell Parameter Phenotype Baseline cells/mm3 Median % Change at C1D15 from Baseline P Baseline cells/mm3 Median % Change at C1D15 from Baseline P Total T cells ABS CD3+ 636.9 17.733 0.24747 522.94 43.83 0.03638 Total T helper ABS CD3+/CD4+/CD8- 275.38 18.333 0.07812 238.96 13.428 0.09893 T helper Activated ABS CD3+/CD4+/CD8-/HLA-DR+ 62.34 105.769 0.00238 57.11 78.571 0.01567 T helper Total Naïve ABS CD3+/CD4+/CD8-/45RA+/45RO- 69.07 -54.545 0.0038 47.94 -47.841 0.03159 T helper Effector CD62L+ ABS CD3+/CD4+/CD8-/45RA+/62L+ 117.62 0 0.16621 93.74 -6 0.14611 T helper Effector CD62L- ABS CD3+/CD4+/CD8-/45RA+/62L- 21.38 -25.862 0.15196 28.44 -20.161 0.08548 T helper Total Memory ABS CD3+/CD4+/CD8-/45RA-/45RO+ 137.93 41.176 0.05373 119.15 36 0.01915 T helper Central Memory ABS CD3+/CD4+/CD8-/45RA-/62L+ 91.9 47.451 0.01953 75.74 37.143 0.01275 T helper Effector Memory ABS CD3+/CD4+/CD8-/45RA-/62L- 44.17 18.147 0.17768 41.07 19.375 0.04749 Total T cytotoxic ABS CD3+/CD4-/CD8+ 334.07 18.044 0.27499 265.7 43.823 0.0127 T cytotoxic Activated ABS CD3+/CD4-/CD8+ /HLA-DR+ 176.76 100 0.20781 121.3 96.454 0.00686 T cytotoxic Total Naïve ABS CD3+/CD4-/CD8+ /45RA+/45RO- 173.69 -35.714 0.15126 116.04 -32.667 0.89774 T cytotoxic Effector CD62L+ ABS CD3+/CD4-/CD8+ /45RA+/62L+ 127.28 20.727 0.24151 93.43 17.419 0.09599 T cytotoxic Effector CD62L- ABS CD3+/CD4-/CD8+ /45RA+/62L- 151.72 -14.286 0.28394 120.98 -18.301 0.18068 T cytotoxic Total Memory ABS CD3+/CD4-/CD8+ /45RA-/45RO+ 55.03 167.402 0.26292 54.13 184.615 0.01034 T cytotoxic Central Memory ABS CD3+/CD4-/CD8+ /45RA-/62L+ 26.83 160.417 0.00013 18.78 264.087 0.00169 T cytotoxic Effector Memory ABS CD3+/CD4-/CD8+ /45RA-/62L- 28.14 133.333 0.00107 32.59 100 0.01939 Table 2. MM n=6-13 NHL n=5-12 Cytokine Baseline cells/mm3 Median % Change from Baseline P Baseline cells/mm3 Median % Change from Baseline P IL-2 98.71 603.509 0.01329 104.5 437.194 0.01761 IL-6 131.84 124.108 0.03426 99.64 21.68 0.2692 GM-CSF 90.24 636.207 0.06608 212.96 144.601 0.16744 IFNg 271.85 404.98 0.0056 554.64 162.451 0.03024 Conclusions: CC-122 significantly increases the proportion of several cytotoxic and helper T cells subsets while decreasing naïve T cells in both DLBCL and MM subjects. CC-122 also significantly activates T cells ex vivo as measured by cytokine production. These results indicate that CC-122 is a potent modulator of T cell numbers and activation and this may serve as rationale for combinations with other immunotherapies. Disclosures Gandhi: Celgene: Employment, Equity Ownership. Off Label Use: CC-122 is a first in class PPM(TM) pleiotropic pathway modifier with multiple biological activities against B lineage cells. Vincent:Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Carpio:Celgene: Research Funding. Stoppa:Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gharibo:Celgene: Research Funding. Damian:Celgene: Research Funding. Rasco:Celgene: Research Funding; Asana BioSciences, LLC: Research Funding. Ysebaert:Celgene: Research Funding. Cordoba:Celgene: Research Funding. Edenfield:Celgene: Research Funding. Pinto:Celgene Corporation: Honoraria; Takeda: Honoraria, Research Funding; Spectrum: Honoraria. López-Martín:Celgene: Research Funding. Sancho:Celgene: Research Funding. Panizo:Janssen: Speakers Bureau; Takeda: Speakers Bureau; Roche: Speakers Bureau; Celgene: Research Funding. Wei:Celgene: Employment, Equity Ownership. Hagner:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership. Hege:Celgene Corporation: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene: Employment.