IKZF3 Protein Research Articles

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Design, synthesis, and biological evaluation of a novel series of 2-(2,6-dioxopiperidin-3-yl)isoquinoline-1,3(2H,4H)-dione derivatives as cereblon modulators

In the current study, we designed and synthesised a novel series of 2-(2,6-dioxopiperidin-3-yl)isoquinoline-1,3(2H,4H)-dione derivatives as cereblon (CRBN) modulators. The results of the CCK8 assay revealed potent antiproliferative activity for the selected compound 10a against NCI-H929 (IC50=2.25 µM) and U239 (IC50=5.86 µM) cell lines. Compound 10a also can inhibit the TNF-α level (IC50=0.76 µM) in LPS stimulated PMBC and showed nearly no toxicity to this normal human cell line. The TR-FRET assay showed compound 10a having potent inhibitory activity against CRBN (IC50=4.83 µM), and the docking study confirmed a nice fitting of 10a into the active sites of CRBN. Further biology studies revealed compound 10a can increase the apoptotic events, arrest the NCI-H929 cells at G0/G1 cell cycle, and induce the ubiquitination degradation of IKZF1 and IKZF3 proteins by CRL4CRBN. These preliminary results suggested that compound 10a could serve as a potential antitumor drug and worthy of further investigation.

Generation of a Lenalidomide-Sensitive Mouse Model for Multiple Myeloma By Expression of the Crbn I391V Mutant in Mopc-315.BM Cells

Lenalidomide is a highly active treatment for multiple myeloma. We and others have previously shown that lenalidomide targets Ikaros (IKZF1) and Aiolos (IKZF3) for ubiquitination and degradation via the cereblon (CRBN) E3 ubiquitin ligase. Depletion of these lymphoid transcription factors selectively inhibits growth of multiple myeloma cells. In contrast to human cells, mouse cells are insensitive to lenalidomide due to a single amino acid change in the murine CRBN (mCRBN) protein (isoleucine instead of valin at position 391). Therefore, lenalidomide could so far not be studied in syngenic mouse multiple myeloma models. The mouse MOPC-315.BM cell line engrafts in the bone marrow of BALB/c mice and induces multiple myeloma after intravenous injection (Riedel et al., PLoS one 2012 and Hofgaard et al., PLoS one 2012). We aimed to generate a lenalidomide-sensitive mouse model by retroviral expression of a mutant murine Crbn where the isoleucine at position 391 is replaced by valin (mCrbnI391V) in MOPC-315.BM cells.Western blot analyses showed that MOPC-315.BM multiple myeloma cells express IKZF1 and IKZF3 proteins at a high level as compared to non-myeloma cell lines. Expression of a dominant negative IKZF3 isoform that inactivates both, IKZF3 and IKZF1, inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on IKZF3 and IKZF1. In unmodified or mCrbn -overexpressing MOPC-315.BM cells even high concentrations (>10 µM) of lenalidomide did not induce degradation of IKZF1 and IKZF3 and consistently had no effect on proliferation. In contrast, we observed effective degradation of IKFZ1 and IKZF3 in MOPC-315.BM expressing mCrbnI391V and these cells were highly sensitive to lenalidomide, pomalidomide, and thalidomide. Having shown that expression of mCrbnI391V sensitizes mouse multiple myeloma cells in vitro, we sought to investigate whether this is also true in vivo .For that purpose, we injected mCrbnI391V-expressing MOPC-315.BM cells in BALB/c mice. Luciferase expression of MOPC-315.BM cells allowed us to monitor tumor growth non-invasively by bioluminescense at different time points. Similar to the unmodified MOPC-315.BM cells, we detected mCrbnI391V-expressing MOPC-315.BM in the bone between days 12 to 19 by bioluminescense and continuous growth of the lesions until mice became sick around day 40. This demonstrated that expression of mCrbnI391V did not affect the potential of MOPC-315.BM to induce multiple myeloma in vivo. We next treated the mice with different dosages and schedules of lenalidomide by intraperitoneal injection when tumor engraftment was clearly measurable by bioluminescense in all mice. Lenalidomide at a high concentration of 25 mg/kg given twice weekly had only weak effects on tumor growth. In contrast, daily treatment with low concentrations (1 mg/KG) effectively inhibited tumor growth, demonstrating that continuous inactivation of IKZF1 and IKZF3 is necessary for lenalidomide activity in vivo .In conclusion, our results demonstrate that mouse multiple myeloma cells can be sensitized to lenalidomide by expression of mCrbnI391V in vitro and in vivo . This model will allow for the first time to test drug combinations including lenalidomide in a syngenic mouse model. Furthermore, it serves as a proof-of-concept for the generation of transgenic lenalidomide-sensitive mouse models for multiple myeloma and other hematologic malignancies in the future. [Display omitted] DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Caspase-8 Inhibition Prevents the Cleavage and Degradation of E3 Ligase Substrate Receptor Cereblon and Potentiates Its Biological Function.

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4), mediates the ubiquitination and degradation of constitutive substrates and immunomodulatory drug-induced neo-substrates including MEIS2, c-Jun, CLC1, IKZF1/3, CK1α, and SALL4. It has been reported that CRBN itself could be degraded through the ubiquitin-proteasome system by its associated or other cullin-RING E3 ligases, thus influencing its biological functions. However, it is unknown whether the CRBN stability and its biological function could be modulated by caspases. In this study, using model cell lines, we found that activation of the death receptor using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) leads to the decreased CRBN protein level. Through pharmacological inhibition and activation of caspase-8 (CASP-8), we disclosed that CASP-8 regulates CRBN cleavage in cell lines. Site mapping experiments revealed that CRBN is cleaved after Asp9 upon CASP-8 activation, resulting in the reduced stability. Using myeloma as a model system, we further revealed that either inhibition or genetic depletion of CASP-8 enhances the anti-myeloma activity of lenalidomide (Len) by impairing CRBN cleavage, leading to the attenuated IKZF1 and IKZF3 protein levels and the reduced viability of myeloma cell lines and primary myeloma cells from patients. The present study discovered that the stability of the substrate receptor of an E3 ligase can be modulated by CASP-8 and suggested that administration of CASP-8 inhibitors enhances the overall effectiveness of Len-based combination therapy in myeloma.

Targeting 1q21 Amplification with APG2575 and Lenalidomide to Sensitize BCL-2 Inhibition with the Decrease of MCL-1 Protein in High Risk MM Models

Background Multiple myeloma (MM) is a heterogenous plasma cell malignancy. About 30-40% of newly diagnosed and 20-60% of relapsed/refractory MM patients carry 1q21 amplification worldwide, a high-risk indicator for poor prognosis in RRMM. Different BCL-2 family anti-death proteins play important roles in MM survival and drug resistance. High expression of BCL-2 due to t (11;14) renders cell vulnerability to BCL-2 antagonist, however, patients carrying other genetic abnormality including 1q21 amplification have limited response to the newly emerged treatment choice. With MCL-1 upregulation accompanied with 1q21 amplification, we tested whether BCL-2 antagonist combined with IMiDs (immunomodulatory imide drugs), improves cell killing in high-risk MM patient models, including those resistant to bortezomib and lenalidomide. For that aim, we studied APG-2575, a novel and potent BCL-2 inhibitor developed by Ascentage Pharma Group and it is currently in clinical trials for hematologic malignances, including MM. Methods 1. Cells were treated with APG-2575 single agent or in combination with lenalidomide; 2. Cell line viability was assessed by CellTiter-Glo (CTG) assay; 3. Flow cytometry analysis was used to detect CD138+ cell surface marker in primary cells derived from MM patients; 4. Western blot analysis for BCL-2 family and IMiD signaling proteins. Results We first determined the cell sensitivity of APG-2575 as a single agent in a panel of MM cell lines, and as expected, those carrying t (11;14) were very sensitive to APG-2575, with low IC50 values ranging 7-23 nM. The IC50 values for cells carrying other genetic markers were greater than 5-10 μM. We then evaluated cell-death inducing activity of APG-2575 in MM patient-derived primary cells ex vivo, which were freshly prepared from patients' bone marrow aspirates. Primary cells were treated with APG-2575 or ABT-199 (venetoclax) for 18-24 hours, and the loss of CD138 surface marker was used to quantify cell death. As shown in Figure 1a, APG-2575 induced cell death (CD138+ loss) in a dose-dependent manner, and significant cell death was observed at 0.37-3.3 μM of APG-2575, indicating primary cells more sensitive than MM cell lines. Interestingly, the sensitivity to APG-2575 is similar in primary MM cells with or without 1q21 amplification. Since moderate cell death inducing activity was observed in MM cells when APG-2575 used as a single agent, we combined APG-2575 with lenalidomide. Cell death was increased in the combination groups compared with single agent, and it was dose-dependent (Figure 1b).Similar enhanced antiproliferative activity was confirmed in RPMI 8226 cell line, which carries 1q21 amplification (Figure 1c). We furthered to understand the mechanism of action (MoA) of this combination. In Figure 1d, Western blot analysis of RPMI 8226 cell line revealed that IKZF1 and IKZF3 proteins from the NF-KB pathway were downregulated by lenalidomide. And the decrease of MCL-1 protein and the strong induction of pro-death protein BAK were evident in the combination group of APG-2575 and lenalidomide, which helps to illustrate MoA underlying the synergistic effect of APG-2575 and lenalidomide in MM models. Conclusions In both cell lines and primary samples derived from MM patients, APG-2575 demonstrates cell death inducing activity as a single agent, and enhanced/synergistic effects when it combines with lenalidomide in RRMM resistant to lenalidomide and bortezomib. The combination decreases IKZF1, IKZF3 and MCL-1 proteins, and upregulates pro-death protein BAK, thus providing a strong rationale to combine BCL-2 inhibitor and lenalidomide to treat high-risk patient populations carrying 1q21 amplification. Disclosures FU: Ascentage Pharma (SuZhou) Co., Ltd: Other: research agreement contract . Yin:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Mao:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Deng:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Fang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.

Development of Label-Free Impedimetric Immunosensors for IKZF1 and IKZF3 Femtomolar Detection for Monitoring Multiple Myeloma Patients Treated with Lenalidomide

Lenalidomide is an immunomodulatory drug (IMiD) used to treat multiple myeloma (MM) patients. Lenalidomide destroys MM cells by inducing ubiquitination and the consequent degradation of Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3). High expression of IKZF1 and IKZF3 in MM results in less sensitivity to lenalidomide treatment and possible cytotoxic effect. Therefore, detecting the expression of IKZF1 and IKZF3 proteins is of utmost importance in the treatment of MM. Here, we report the fabrication of a novel label-free electrochemical immunosensor for the rapid detection and quantification of IKZF1 and IKZF3 using electrochemical impedance spectroscopy (EIS). Gold electrodes were used to fabricate the immunosensors by immobilizing IKZF1 and IKZF3 specific antibodies using cysteamine and PDITC crosslinkers. The immunosensors were able to detect IKZF1 and IKZF3 protein levels with respective low detection limits of 0.68 and 0.97 pg/ml (11.8 and 16.7 fM). Furthermore, the immunosensors’ successful application in human serum and their high selectivity and sensitivity enables their possible application in other biofluids as simple point-of-care devices for monitoring multiple myeloma patients treated with lenalidomide, to prevent the drug’s cytotoxicity and minimize its side effects.

Genome-Wide Analysis of BRD4-Targets Reveals the Therapeutic Relevance of Simultaneous Targeting of BCR Pathway and IKZF-MYC Axis in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. DisclosuresKuroda:Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

Molecular Mechanism of CRBN in the Activity of Lenalidomid eagainst Myeloma--Review

Cereblon（CRBN) is a brain-associated protein with ionic protease activity, which interacts with DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B), and regulator of Cullins 1 (RoC1) to form the functional E3 ubiquitin ligase complex(CRBN-CRL4) that performs proteolysis via the ubiquitin-proteasome pathway. And CRBN is a necessary target protein for the anti-myeloma effect of immunomodulators. The combination of lenalidomide and CRBN recruited a new substrate that binds to the CRBN-CRL4 complex, leading to increased ubiquitination and proteasome-dependent degradation, thus resulting in anti-myeloma activity. The substrates binding to this complex are IKZF1, IKZF3 proteins and GS, etc. The CRBN-dependent degradation of IKZF1 and IKZF3 after lenalidomide treatment is also the result of H2O2-mediated oxidative stress. In addition to ubiquitination, lenalidomide also mediates ubiquitin-independent pathways that prevent CRBN from binding to CD147-MCT1 in a competitive manner to regulate its antitumor activity. Lenalidomide can also play a role in multiple myeloma(MM) cells by modulating miRNA levels and CRBN binding to downstream protein AGO2 expression. Thus, there are many molecular mechanisms of lenalidomide anti-myeloma activity. This review summarizes the molecular mechanisms of CRBN in lenalidomide against myeloma activity in terms of ubiquitin-dependent and ubiquitin-independent pathways.

Fate of Ikaros in Multiple Myeloma Cells Upon Treatment With Lenalidomide and Proteasome Inhibitor

Abstract Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Download : Download high-res image (188KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Fate of Ikaros in Multiple Myeloma Cells upon Treatment with Lenalidomide and Proteasome Inhibitor

Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells.Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c).Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IKZF1/3 Protein Expressions Are Associated with a Better Survival in Relapsed/Refractory Multiple Myeloma Patients Treated with Lenalidomide

It has been demonstrated that lenalidomide causes selective degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos) which are two essential transcription factors for proliferation of multiple myeloma cells. Consequently, the drug sets up a molecular sequence of events that lead to programmed cell death in the tumoral cells. This anti-proliferative effect is mediated by down-regulation of c-Myc and interferon regulatory factor 4 (IRF4). However, it is not clear whether IKZF1/IKZF3 protein expression in myeloma cells is predictive of clinical outcome. Thus, we evaluated bone marrow samples of 50 relapsed/refractory multiple myeloma (MM) patients regarding IKZF1/3 protein expression before starting lenalidomide. There were 31 males and 19 females with median age of 59 years (range 41-75). They all had received lenalidomide-based therapy after relapse following autologous stem cell transplantation (ASCT), thalidomide, or bortezomib. The median follow-up was 86.4 months. By immunohistochemistry (IHC), CD138 positive myeloma cell aggregates were examined for IKZF1 and IKZF3 protein expression. We used H-score method (range 0-300) based on the intensity and percentage of the stained myeloma cells. Cases were considered positive for IKZF1 or IKZF3 if H-score is equal or over 150 or 200, respectively. IKZF1 showed nuclear staining but IKZF3 showed both nuclear and cytoplasmic staining.IKZF1 and IKZF3 were expressed in 72% and 58% of the bone marrow specimens, respectively. IKZF1 and IKZF3 expressions were strongly correlated (p<0.0001). Both IKZF1 & IKZF3 expressions were associated with longer progression free (p=0.0029 & p<0.0001, respectively) and overall survivals (p=0.0014 & p<0.0001, respectively). IKZF3 (p=0.0025) expression but not IKZF1 (p=0.094) was correlated with the clinical response to lenalidomide. There was no significant association between IKZF1/3 protein expression and other clinical or biological risk factors including age, gender, International staging system (ISS), hemoglobin, calcium, creatinine, bone lytic lesions, β2 micro-globulin, albumin or cytogenetic risk factors such as del (13q), del (17p), t(4;14), and amp (1q21).Our study demonstrates that expressions of the IKZF1/3 proteins detected by IHC are correlated with superior survival outcome in refractory MM patients treated with lenalidomide. IHC is routinely available, robust and inexpensive method. Thus, if confirmed in a larger prospective study, IKZF1/3 immunostaining can be readily adopted in clinical practice for prediction of drug response and clinical outcomes in MM patients receiving lenalidomide therapy. DisclosuresReece:Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Merck: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

SYK-Inhibitor Bay 61-3606 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells Independent of SYK Inhibitory Effects but Via Degradation of IKZF1/3

Introduction: Bay 61-3606 is a cell-permeable imidazopyrimidine compound that acts as a potent, ATP-competitive, reversible, and highly selective inhibitor of Syk tyrosine kinase activity with no inhibitory effect against Btk, Fyn, Itk, Lyn, and Src. BAY 61-3606 has been also shown to inhibit Syk-mediated cellular functions such as glucose-tyrosine phosphorylation of I κ B α and p65 nuclear translocation. It further exhibits a good oral bioavailability and in vivo efficacy in rat models. Recently, Bay 61-3606 was found to inhibit cell proliferation and SDF-1a-induced migration of MM cells (1). Based on these promising preliminary data we further investigated the potential of Bay 61-3606 as new anti-MM agent.Methods and Results: Bay 61-3606, at concentrations as low as 10 nM, induced significant (p<0.01) inhibition of cell growth in MM cells (MM.1S, H929 and RPMI-8266) as shown by WST-1 cell proliferation assay. The inhibition of proliferation was accompanied by increased cell cycle arrest at G0/G1 (from 60% to 75%). More importantly, Bay 61-3606 dose dependently induced MM cells apoptosis suggesting that Bay 61-3606 has potent anti-MM properties. Since Bay 61-3606 was originally reported as a SYK inhibitor, we analyzed whether the effects of Bay 61-3606 were due to SYK inhibition. For this purpose, we first checked the expression of SYK in all the MM cell lines (MM.1S, H929, U266, OPM2 and RPMI-8226). And to our surprise, although all these cells were sensitive to Bay 61-3606 treatment, several of these cell lines (H929, U266 and RPMI-8226) had no detectable SYK expression by Western Blot assay. This suggests that the anti-MM effects of Bay 61-3606 are not mediated by SYK inhibition, which was further confirmed by using another specific SYK inhibitor GS-9973. Further mechanistic studies showed that Bay 61-3606 significantly downregulated protein levels of IKZF1, IKZF3, c-MYC and IRF-4 in IMiD®-sensitive and resistant MM cell lines (MM.1S, H929 and RPMI-8266) by Western Blot assay. In contrast, real-time PCR assay indicated IKZF1/3-mRNA level was not altered, suggesting that Bay 61-3606 regulates IKZF1/3 at post-translational level. The proteinase inhibitor MG132 and cullin-dependent ubiquitin ligase inhibitor MLN4924 blocked the downregulation IKZF1/3 by Bay 61-3606 confirming that Bay 61-3606 induced IKZF1/3 protein degradation. Consistent with its proapoptotic effects, Bay 61-3606 downregulated MDM2 phosphorylation, upregulated P53 level and induced PARP cleavage as confirmed by Western Blot assy.Conclusion: In summary, our results demonstrate that Bay 61-3606 has anti-MM effects by inducing cell cycle arrest and apoptosis, and this effect is independent of SYK inhibition. Similar to IMiD® compounds, Bay 61-3606 induces IKZF1 and IKZF3 protein degradation in MM cells. More importantly, Bay 61-3606 has effects on both IMiD®-sensitive and resistant myeloma cells and may represent a novel potent anti-MM agent.

Inhibition of CDK4/CDK6 Sensitizes Myeloma to IMiD By Reducing the MEIS2 to Cereblon Ratio That Accelerates IKZF1 and IKZF3 Degradation

Lenalidomide (Len) and pomalidomide (Pom) are immunomodulatory drugs (IMiDs) effective in hematologic malignancies, in combination therapies for multiple myeloma (MM) in particular. Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Emerging evidence suggests that IMiDs bind CRBN and block an endogenous substrate MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN and their degradation. This then leads to loss of IRF4 necessary for myeloma survival. The clinical relevance of these novel findings, however, has not been defined.To address this question, we've investigated the mechanism of IMiD action and the functional consequences in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=31) in stromal co-culture ex vivo in the context of the clinical response to Len or Pom in vivo before or after biopsy. We showed by whole transcriptome sequencing, protein analysis and functional assays that 1) BMMCs are addicted to IKZF3-IRF4 for survival; 2) Len-mediated IRF4 loss leads to de-repression of IRF7, induction of interferon (IFN) response genes and TRAIL-mediated apoptosis; and 3) the magnitude of IFN induction is tightly associated with killing of BMMCs by Len. Importantly, the IMiD sensitivity in BMMCs ex vivo correlated with the prior or subsequent clinical response to IMiD-based therapies in individual myeloma patients, suggesting that the clinical response to IMiDs in myeloma is largely intrinsic to myeloma cells.IMiDs have been reported to cause cell cycle arrest. We found that before evidence of killing, Len and Pom induced late G1 arrest by both repressing CCNA2 (encoding cyclin A) mRNA synthesis and elevating p21 and p27 proteins independent of Rb and p53. This result suggests that IMiDs preferentially kill cells in G1 arrest, and that induction of p rolonged early G1 arrest (pG1) beyond the normal G1 transit time by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991, Ibrance) may sensitize MM cells to IMiD killing, as it does to killing by other agents. Indeed, induction of pG1 by palbociclib overrides cell cycle regulation by Len, and sensitizes BMMCs to Len-mediated apoptosis by augmenting the loss of IRF4 protein and the induction of IRF7, IFNb and TRAIL.Further investigation revealed that induction of pG1 by CDK4/CDK6 inhibition sensitizes primary myeloma cells to IMiD killing by rapid acceleration of Len-mediated loss of IKZF1 and IKZF3 proteins, within one hour of IMiD addition. Loss and gain of function studies demonstrates that MEIS2 opposes pG1 sensitization to Len killing; however, MEIS2 itself is regulated by the cell cycle. Induction of pG1 reduces the ratio of MEIS2 to CRBN by both reducing the MEIS2 protein rapidly and increasing the CRBN protein at a later time in cooperation with Len.In summary, our data provide the first evidence that induction of prolonged early G1 arrest by selective inhibition of CDK4/CDK6 amplifies IMiD killing of primary myeloma cells by both repressing MEIS2 and increasing CRBN protein in cooperation with Len. This leads to a profound reduction in the ratio of MEIS2 to CRBN that accelerates the loss of IKZF1, IKZF3 and IRF4, and enhances IFN and TRAIL induction. Reducing the MEIS2/CRBN ratio thus represents a novel mechanism by which CDK4/CDK6 inhibition sensitizes myeloma to IMiDs, and a means for developing mechanism-based IMiD therapy through cell cycle control. DisclosuresHuang:Celgene: Research Funding. Off Label Use: Palbociclib (PD 0332991) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Rossi:Calgene: Speakers Bureau. Pearse:Celegen: Consultancy. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Chen-Kiang:Celgene: Consultancy.

IKZF3 p.L162R Is a Recurrent Hotspot Mutation in Chronic Lymphocytic Leukemia (CLL)

Abstract Recent advances in massively parallel DNA sequencing has enabled identification of various somatic driver mutations in chronic lymphocytic leukemia (CLL). However, recent study has shown that the search of recurrent driver mutations are far from complete, and sequencing of extended cohort continues to reveal rare but recurrent driver mutations. To search for rare but biologically relevant driver mutations, we conducted whole exome sequencing (WES) on 54 bone marrow samples from patients with CLL, 35 (60%) of whom were untreated and 19 (40%) relapsed after chemoimmunotherapy such as FCR or BR regimens. WES achieved mean targeted sequencing coverage of 124x. Somatic single nucleotide variant (SNV) and small insertions/deletions were called using Mutect and Pindel algorithm, respectively, against matched normal control. Table 1 shows the comparison of clinical characteristics between untreated and treated patients. As expected, patients previously treated with chemo-immunotherapy had more advanced clinical features compared to untreated patients, such as older age, more advanced Rai stage, higher incidence of 17p deletion, higher levels of serum beta 2 microglobulin, and worse thrombocytopenia. The median number of somatic SNV was 29 (range: 14-59) per case. There was no significant difference in the numbers of somatic SNV between untreated and treated patients (median number of SNV: 27 [range: 12-59] in untreated vs. 32 [range: 14-59] in treated, P = 0.913). There was no association between the number of SNV and other clinical features such as Rai stage, IGHV mutation status, and 17p deletion status, although analyses are limited by patient number. Mutation signature was also similar between untreated and treated patients. In addition to previously reported recurrent mutations in CLL, 3 patients (6%) had somatic hot-spot mutation in IKZF3, which was a heterozygous missense mutation (T&gt;G) resulted in p.L162R. Two of the 3 patients with IKZF3 mutation were previously treated and the mutation was clonal by ABSOLUTE analysis (cancer cell fraction &gt; 0.9). Both patients had high risk disease features such as deletion 17p and ZAP70 positivity. The other treatment-naive patient had subclonal IKZF3 mutation. One patient with clonal IKZF3 mutation responded well to IPI-145 (PI3K delta gamma inhibitor). IKZF3 is a transcription factor, which plays an important role in B-cell differentiation, proliferation and maturation. It has three zinc finger domains, the L162R mutation resides in the second zinc finger domain. The mutated residue was highly conserved among all species. In the COSMIC database, IKZF3 p.L162R somatic mutation was also found in diffuse large B cell lymphoma and mantle cell lymphoma, suggesting shared genetic mechanisms among other B-cell ,malignancies. In a recent study, IKZF3 p.L162R was shown to inhibit ubiquitination of IKZF3 protein by lenalidomide therapy in 293T cell (Kršnke et al. Science 2014). Therefore, we tested expression of IKZF3 protein in CLL bone marrow by immunohistochemistry. Very strong nuclear expression of IKZF3 was observed in the IKZF3 mutated cases whereas IKZF3 expression was weak in wild type cases, suggesting that p.L162R mutation leads to longer-lived IKZF3 protein. We are currently sequencing bone marrow samples from CLL patients treated with lenalidomide to evaluate association between IKZF3 mutation and response to lenalidomide therapy. Table 1. Characteristics of 54 CLL patients who underwent bone marrow WES. Characteristic Untreated Treated P n = 35 n = 19 Median age (range), y 61 (37-84) 69 (56-81) 0.009 Female Rai Stage (%) 0.131 0 12 (34) 2 (11) I 9 (26) 4 (21) II 2 (6) 1 (5) III 5 (14) 2 (11) IV 7 (20) 10 (53) Rai Stage 0-II (%) 23 (66) 7 (37) 0.041 Rai Stage III-IV (%) 12 (34) 12 (63) Deletion 17p (%) 4 (11) 7 (37) 0.027 Complex karyotype (%) 4 (12) 4 (24) 0.243 IGHV mutation status (%) Mutated 15 (43) 7 (37) 0.546 Unmutated 10 (19) 7 (37) Unknown 10 (19) 5 (26) ZAP70 status (%) Negative 16 (46) 6 (32) 0.313 Positive 19 (54) 13 (68) Median WBC count (range), x 109/L 25.2 (4.3-140.8) 12.5 (2.5-161.7) 0.163 Median HGB count (range), g/dL 13.6 (7.4-16.5) 12.3 (8.5-16.1) 0.101 Median BM CLL cells by flow (range), % 71 (22-91) 76 (0.8-96) 0.825 Serum B2MG (range), mg/dL 1.69 (0.9-7.0) 4.4 (2.2-12.4) 0.001 Disclosures Burger: Pharmacyclics LLC, an AbbVie Company: Research Funding. Keating:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Wierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding.

The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma.

Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells.Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells.

Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3

Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy. We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells.To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment. Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN's role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide's effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate.Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation.IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells.Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide.In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide's selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures:Ebert:Celgene: Membership on an entity's Board of Directors or advisory committees.

The IKZF3 (Aiolos) transcription factor is highly upregulated and inversely correlated with clinical progression in chronic lymphocytic leukaemia

The IKZF3 (Aiolos) transcription factor is highly upregulated and inversely correlated with clinical progression in chronic lymphocytic leukaemia