Ikaros Family Zinc Finger Protein Research Articles

Mezigdomide-A Novel Cereblon E3 Ligase Modulator under Investigation in Relapsed/Refractory Multiple Myeloma.

Mezigomide is an oral cereblon E3 ligase modulator (CELMoD) that is under clinical investigation in patients with relapsed/refractory (RR) multiple myeloma (MM). Like other CELMoD compounds, mezigdomide acts by altering the conformation of cereblon within the cullin 4A ring ligase-cereblon (CRL4CRBN) E3 ubiquitin ligase complex, thereby recruiting novel protein substrates for selective proteasomal degradation. These include two critical lymphoid transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), also known as Ikaros and Aiolos, which have important roles in the development and differentiation of hematopoietic cells, in MM pathobiology, and in suppressing the expression of interferon-stimulating genes and T-cell stimulation. Among the CELMoDs, mezigdomide has the greatest cereblon-binding potency, plus the greatest potency for the degradation of Ikaros and Aiolos and subsequent downstream antimyeloma effects. Preclinical studies of mezigdomide have demonstrated its anti-proliferative and apoptotic effects in MM, along with its immune-stimulatory effects and its synergistic activity with other antimyeloma agents, including in lenalidomide-/pomalidomide-resistant MM cell lines and mouse xenograft models. Early-phase clinical trial data indicate notable activity in heavily pretreated patients with RRMM, including those with triple-class-refractory disease, together with a tolerable and manageable safety profile. This review summarizes current preclinical and clinical findings with mezigdomide and its potential future roles in the treatment of MM.

CFT7455, a novel IKZF1/3 degrader, demonstrates potent activity in peripheral and CNS models of NHL as a single agent or in combination with clinically approved agents

Immunomodulatory drugs, such as pomalidomide and lenalidomide are approved for the treatment of multiple subtypes of NHL. These drugs induce an interaction between Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) and cereblon (CRBN), an E3 ligase, resulting in IKZF1/3 degradation. Clinically, immunomodulatory drugs are active as single agents or when used in combination with several classes of targeted therapies, in both first line and relapsed/refractory NHL subtypes. We previously described the preclinical characterization of CFT7455 as a novel IKZF1/3 degrader with 800-fold increased potency over pomalidomide in CRBN binding. We demonstrated that this increased activity translates into dramatically greater efficacy compared to pomalidomide in cellular and in vivo preclinical models of multiple myeloma and NHL. Here we show that the increased catalytic activity and improved potency of CFT7455 also translates into potent anti-tumor activity as a single agent or in combination with clinically approved agents in multiple in vivo models of NHL. In addition, the enhanced activity of CFT7455 demonstrates CNS activity in both the Raji and OCI-Ly10 intracranial xenograft models. CFT7455 (100 µg/kg/day) led to a significant increase in survival probability in comparison to pomalidomide (3000 µg/kg/day) and two investigational IKZF1/3 degraders in both models. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Aggressive T-cell non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. M. J. Thomenius Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. Perino Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. Kirby Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics R. Agafonov Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics P. Chaturvedi Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. Eron Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics A. Good Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. A. Hart Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics M. He Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics A. Phillips Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics D. A. Proia Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics J. A. Henderson Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics C. Nasveschuk Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics S. L. Fisher Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics R. M. Pollock Employment or leadership position: C4 Therapeutics Stock ownership: C4 Therapeutics

CFT7455, a Novel IKZF1/3 Degrader, Demonstrates Potent Anti-Tumor Activity in Models of Non-Hodgkin's Lymphoma As a Single Agent or in Combination with Clinically Approved Agents

Immunomodulatory drugs, such as pomalidomide and lenalidomide are approved for the treatment of multiple subtypes of non-Hodgkin's lymphoma (NHL). These drugs induce an interaction between Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) and cereblon (CRBN), an E3 ligase, resulting in IKZF1/3 degradation. IKZF1/3 are master transcription factors that play a key role in B and T-cell differentiation and maintain levels of interferon regulatory factor 4 (IRF4), a critical regulator in NHL. Clinically, immunomodulatory drugs have shown activity as single agents and when used in combination with several classes of targeted therapies, such as rituximab, in both first line and relapsed/refractory NHL subtypes. We previously described the preclinical characterization of CFT7455 as a novel IKZF1/3 degrader with 800-1600-fold increased potency over pomalidomide in CRBN binding and cellular IKZF1 degradation assays. We demonstrated that this increased activity translated into dramatically greater efficacy compared to pomalidomide in cellular and in vivo preclinical models of multiple myeloma and NHL. Here we show that the increased catalytic activity and improved potency of CFT7455 also translates into potent anti-tumor activity as single agent or in combination with clinically approved agents in central nervous system (CNS) and subcutaneous models of NHL. In both the Raji and OCI-Ly10 CNS xenograft models, CFT7455 (100 µg/kg/day) led to a significant increase in survival in comparison to pomalidomide (P=0.002, P=0.0002). The combination of immunomodulatory drugs with CD20, BTK, or HDAC targeting agents shows promising clinical activity in the treatment of NHL. Combination studies were conducted, dosing CFT7455 in combination with rituximab, ibrutinib, or the HDAC inhibitor romidepsin in several models of NHL. In the mantle cell lymphoma (MCL) model, Mino, the combination of CFT7455 with rituximab demonstrated synergistic activity and complete tumor regression. In the diffuse large B-cell lymphoma (DLBCL) model, TMD8, the combination of ibrutinib and CFT7455 was synergistic and resulted in a significant increase in survival probability. In the CNS NHL model, OCI-Ly10, the combination of CFT7455 with rituximab or ibrutinib also led to a significant increase in survival probability highlighting the therapeutic potential of this combination. Synergistic activity was also observed with the combination of CFT7455 and romidepsin in two models of anaplastic large cell lymphoma (ALCL). In conclusion, CFT7455 is a potent, selective catalytic degrader of IKZF1/3, with single agent and combination antitumor activity in DLBCL, ALCL, and MCL models, supporting clinical investigation of CFT7455 for NHL.

Abstract CT186: Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM

Abstract Introduction: CFT7455 is a highly potent and novel Ikaros Family Zinc Finger Protein 1/3 (IKZF1/3) degrader. In xenograft models, CFT7455 has more potent IKZF1/3 degradation compared to other degraders. Early observations from the FIH clinical trial (NCT04756726) along with supporting translational studies are presented here. Methods: Pre-clinical studies comparing CFT7455 and CC-92480 in both in vitro and xenograft models were performed. The pre-clinical studies’ results coincided with observations from the on-going clinical trial. The clinical trial is an open-label, Phase 1/2, multi-center, FIH study in heavily pretreated relapsed/refractory (R/R) MM and non-Hodgkin’s lymphoma (NHL) patients evaluating safety, tolerability, and PK of CFT7455. Eligible MM patients are R/R to therapy and are not candidates for regimens known to provide clinical benefit. A starting dose of 50 μg QD 21 days on/7 days off (21/7) in 28-day cycles was administered. Results: CFT7455 and CC-92480 showed similar cereblon binding profiles and in vitro IKZF1/3 degradation kinetics, translating into sub-nanomolar GI50 values in proliferation assays across a panel of MM cell lines. In the NCI-H929 xenograft model, 100 μg/kg/day of CFT7455 resulted in durable tumor regressions, while 1000 μg/kg/day of CC-92480 gave tumor stasis. Similar results were seen in a systemic model of MM, MM1.S. Both compounds achieved >95% IKZF3 degradation in tumors 4h post dose. At 48h post dose, CFT7455 was more effective than CC-92480 in maintaining IKZF3 degradation (65% vs. 6% respectively). When levels of CFT7455 and CC-92480 in plasma and tumor were compared, CFT7455 concentrations were > DC80 in tumor 48h post dose, while CC-92480 levels were undetectable in tumor and plasma, demonstrating CFT7455 has longer exposure resulting in sustained IKZF1/3 degradation in pre-clinical models. In cohort A, 5 heavily pre-treated MM patients (pts) received single agent CFT7455. 4 pts have received at least 3 cycles, with 2 pts receiving 5 cycles. Neutropenia (grade 4) was observed in 3/5 pts without coincident fever or infection. Additionally, a 2-4 fold accumulation in plasma CFT7455 exposure at steady state was observed. Early pharmacodynamic (PD) data demonstrates deep persistent degradation of IKZF3 (~100%) and serum free light chain reduction (up to 72%) in response to treatment. Stable disease has been observed in 34 pts, suggesting clinical benefit. Conclusions: While CFT7455 showed clinical benefit at 50 ug with deep target degradation, neutropenia was dose limiting. PK/PD modeling suggests alternative dosing regimens may result in increased tolerability with preserved efficacy, and evaluation of them is underway. Updated results will be presented at the meeting. Citation Format: Sagar Lonial, Shambavi Richard, Jeffrey V Matous, Andrew J. Yee, Urvi Shah, Neha Mehta-Shah, Thomas Martin, Eli Muchtar, Sikander Ailawadhi, Paul G. Richardson, Manisha Bhutani, Samantha Perino, Jason Kirby, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, Matthew Schnaderbeck, Michael R. Palmer, Cathleen Gorman, Oliver Schoenborn-Kellenberger, Amanda Hoerres, Stewart L. Fisher, Roy M. Pollock, Adam Crystal, Michelle Mahler, Jesus Bardeja. Pharmacokinetic (PK) profile of a novel IKZF1/3 degrader, CFT7455, enables significant potency advantage over other IKZF1/3 degraders in models of multiple myeloma (MM) and the results of the initial treatment cohort from a first-in-human (FIH) phase 1/2 study of CFT7455 in MM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT186.

Abstract ND13: The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma

Abstract Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Novel Molecular Mechanism of Lenalidomide in Myeloid Malignancies Independent of Deletion of Chromosome 5q.

Simple SummaryLenalidomide is an immunomodulatory drug (IMiD) that has achieved clinical efficacies in multiple myeloma (MM) and myelodysplastic syndromes (MDS) with a single deletion of chromosome 5q (del(5q)). However, many patients treated with lenalidomide relapse and become resistant. Recent studies have demonstrated that lenalidomide binds a protein called cereblon (CRBN), leading to reduced protein levels of IKZF1 and IKZF3 and casein kinase 1 alpha. We have identified signaling molecules downstream of IKZF1, G protein-coupled receptor 68 (GPR68) and regulator of calcineurin 1 (RCAN1) in myeloid malignancies, including MDS and acute myeloid leukemia (AML) with or without del(5q). This review summarizes how lenalidomide exerts anti-tumor activity and highlights novel therapeutic targets that could enhance the anti-tumor activity of lenalidomide with a focus on myeloid malignancies, especially without del(5q).Lenalidomide as well as other immunomodulatory drugs (IMiDs) have achieved clinical efficacies in certain sub-types of hematologic malignancies, such as multiple myeloma, lower-risk myelodysplastic syndromes (MDS) with a single deletion of chromosome 5q (del(5q)) and others. Despite superior clinical response to lenalidomide in hematologic malignancies, relapse and resistance remains a problem in IMiD-based therapy. The last ten years have witnessed the discovery of novel molecular mechanism of IMiD-based anti-tumor therapy. IMiDs bind human cereblon (CRBN), the substrate receptor of the CRL4 E3 ubiquitin ligase complex. Binding of CRBN with IMiDs leads to degradation of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) and casein kinase 1 alpha. We have found that lenalidomide-mediated degradation of IKZF1 leads to activation of the G protein-coupled receptor 68 (GPR68)/calcium/calpain pro-apoptotic pathway and inhibition of the regulator of calcineurin 1 (RCAN1)/calcineurin pro-survival pathway in MDS and acute myeloid leukemia (AML). Calcineurin inhibitor Cyclosporin-A potentiates the anti-leukemia activity of lenalidomide in MDS/AML with or without del(5q). These findings broaden the therapeutic potential of IMiDs. This review summarizes novel molecular mechanism of lenalidomide in myeloid malignancies, especially without del(5q), in the hope to highlight novel therapeutic targets.

Caspase-8 Regulates the Antimyeloma Activity of Bortezomib and Lenalidomide.

Proteasome inhibitors and immunomodulatory drugs (IMiDs) are two major types of drugs for the treatment of multiple myeloma. Although different combination therapies for myeloma have been developed and achieved high responsive rate, these strategies frequently result in drug resistance. Therefore, it is necessary to explore new molecular mechanisms and therapeutic approaches to fulfill this unmet medical need. Here, we find that proteasome inhibitor bortezomib (Btz) causes cereblon (CRBN) cleavage and that caspase-8 (CASP-8) is responsible for this cleavage. Either inhibition or genetic depletion of CASP-8 decreased the CRBN cleavage upon Btz treatment, which could potentiate the antimyeloma activity of IMiD lenalidomide (Len). This work suggests that administration of CASP-8 inhibitors might enhance the overall effectiveness of Btz/Len-based therapeutic treatment of patients with myeloma. SIGNIFICANCE STATEMENT: Caspase-8 activation upon bortezomib treatment results in the cleavage of cereblon, a substrate receptor of the cullin-4 RING E3 ligase, which is responsible for the degradation of two transcription factors, Ikaros family zinc finger protein (IKZF) 1 and IKZF3, in the presence of immunomodulatory drugs including lenalidomide. The administration of caspase-8 inhibitor may enhance the antimyeloma activity of the combination therapy with bortezomib and lenalidomide.

Hsp90/C terminal Hsc70-interacting protein regulates the stability of Ikaros in acute myeloid leukemia cells.

The stability of Ikaros family zinc finger protein 1 (Ikaros), a critical hematopoietic transcription factor, can be regulated by cereblon (CRBN) ubiquitin ligase stimulated by immunomodulatory drugs in multiple myeloma. However, other stabilization mechanisms of Ikaros have yet to be elucidated. In this study, we show that the pharmacologic inhibition or knockdown of Hsp90 downregulates Ikaros in acute myeloid leukemia (AML) cells. Proteasome inhibitor MG132 but not autophagy inhibitor chloroquine could suppress the Hsp90 inhibitor STA-9090-induced reduction of Ikaros, which is accompanied with the increased ubiquitination of Ikaros. Moreover, Ikaros interacts with E3 ubiquitin-ligase C terminal Hsc70 binding protein (CHIP), which mediates the STA-9090-induced ubiquitination of Ikaros. In addition, the knockdown of Ikaros effectively inhibits the proliferation of leukemia cells, but this phenomenon could be rescued by Ikaros overexpression. Collectively, our findings indicate that the interplay between HSP90 and CHIP regulates the stability of Ikaros in AML cells, which provides a novel strategy for AML treatment through targeting the HSP90/Ikaros/CHIP axis.

Malignant Clonal Cell Proliferation in Multiple Myeloma and the Hypercoagulable State

Introduction: The malignant clonal cell proliferation in multiple myeloma (MM) results in significant immune dysregulation through clonal specific T cell expansion, elevated levels of CD4+ CD25+FOXP3+ T regulator cells, downregulation of NK cells, high levels of IL-6 and activation of immunosuppressive tumour associated macrophages (TAM) . However, the mechanisms underlying the hypercoagulable state in MM and predisposing to venous thromboembolic (VTE) complications is unclear. Confounding disease factors is the use of immunomodulatory drugs (IMiDs) such as Lenalidomide, Thalidomide and Pomalidomide causing ubiquitination and degradation of the Ikaros Family Zinc Finger Protein (IKZF)1 and 3 by cereblon which also contributes to the prothrombotic effect despite thromboprophylaxis. Aims: The aim of this study was to analyse the changes in the coagulation profile and plasma cell disease burden with treatment, including Lenalidomide, in patients with MM to help define potential underlying mechanisms for hypercoagulability and thrombosis. Methods: Coagulation profiles and disease markers were retrospectively analysed in 16 MM patients receiving treatment with Daratumumab, Lenalidomide and Dexamethasone (DRd) at The Alfred Hospital, Melbourne from April 2019 to August 2020. Patients enrolled were transplant eligible with MM that was refractory to initial induction therapy with Velcade, Cyclophosphamide and Dexamethasone (VCD). This study was approved by The Alfred Hospital ethics committee. Statistical analysis was performed using descriptive statistics and the Wilcoxon Sign Rank Test to compare the median coagulation profiles and disease markers after 1-2 cycles of DRd and 3-4 cycles of DRd. Biomarkers included Prothrombin Time (PT), Partial Thromboplastin Time (PTT), Fibrinogen, Thrombin Clotting Time (TCT), serum paraprotein (SPEP) and serum free light chain (SFLC) with a p value of <0.05 indicating statistical significance. Results: A total 7 patients had coagulation profiles at the two time points i) post 1-2 cycles and ii) post 3-4 cycles of DRd (Table 1). 9 patients had coagulation profiles only after 3-4 cycles of DRd. A separate analysis was performed using Wilcoxon Sign Rank with imputed median differences to allow the inclusion of the 9 additional patients subsequently increasing the sample size to a total of 16 (Table 2). All patients were on anticoagulation with aspirin (n=14), rivaroxaban (n=1) or clopidogrel (n=1) at the time of the analysis. The analysis showed a statistically significant reduction in PT from median 13.6s (11.9-16.6) to 12.7s (11.7-14.1) with 3-4 cycles DRd in both analysis (p=0.006 and p= 0.027). Fibrinogen levels reduced from median of 5.4 g/L (2.8-8) to 3.98 g/L (2.3- 5.1) after 3-4 cycles (p=0.001). TCT increased after 3-4 cycles of DRd (p=0.005). Serum paraprotein demonstrated statistically significant reduction from 10.8g/L (6 -13) to 7.6 g/L (2-13) after 3-4 cycles (p=0.007). Serum free light chain assay also demonstrated reduction in median values from 82.8mg/L (8.1 - 415.7) to 54.6mg/L (0.8 - 338.6) but was not statistically significant (p=0.084). Discussion: All the patients in this study either responded to treatment or had stable disease after treatment with DRd. The data demonstrate a coagulation response to treatment. The median fibrinogen level that was above the upper limit of normal declined on treatment, the TCT increased and the PT decreased, all coinciding with the statistically significant decline in paraprotein level. A larger study is required to confirm these findings. However, this study has demonstrated that the hypercoagulable state in MM improves with disease response. Disclosures Spencer: Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Pharmamar: Other; Secura Bio: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Servier: Consultancy, Other: Grant/Research Support; Janssen: Consultancy, Honoraria, Other: Grant/Research Support, Speakers Bureau; Haemalogix: Consultancy, Honoraria, Other: Grant/Research Support; BMS: Honoraria, Other: Grant/Research Support, Research Funding, Speakers Bureau; TheraMyc: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Grant/Research Support; Takeda: Honoraria, Other, Speakers Bureau; Antegene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Grant/Research Support.

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.

RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation, and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

Cereblon-binding proteins expression levels correlate with hyperdiploidy in newly diagnosed multiple myeloma patients

Immunomodulatory drugs (IMIDs) are very effective in the treatment of multiple myeloma (MM). The description of their cereblon-mediated mechanism of action was a hallmark in MM research. Although the importance of IMID-induced degradation of cereblon-binding proteins is well described in vitro, the prognostic value of their expression levels in MM cells is less clear. Based on recently published data showing somewhat conflicting RNA levels, we analyzed the association between the levels of the Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and karyopherin subunit alpha 2 (KPNA2) proteins measured by flow cytometry and prognostic parameters in 214 newly diagnosed MM patients who were randomized in the GMMG HD6 trial. No statistically significant associations between the expression levels and age, gender, light chain type, International Staging System (ISS) stage or cytogenetic high- and normal risk groups could be identified. Hyperdiploid MM cells expressed significantly higher levels of IKZF1, IKZF3 and KPNA2 than nonhyperdiploid cells. In contrast, translocation t(11;14) was associated with significantly lower expression levels. In conclusion, the observed overexpression of cereblon-binding proteins in MM cells with gain of chromosomes 5, 9, 11, 15, and 19 is consistent with the previously proposed positive regulation of MYC by IKZF1 and IKZF3, as well as MYC activation in hyperdiploid MM cells.

Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity

AbstractLenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment of multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q [del(5q) MDS]. To identify the cellular machinery required for lenalidomide-induced CRL4CRBN activity, we performed a positive selection, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) screen in a lenalidomide-sensitive myeloma cell line. CRBN was the top-ranking gene, with all CRBN-targeting guide RNAs (gRNAs) ranking as the 6 highest-scoring gRNAs. A counterscreen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and 2 E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the constitutive photomorphogenesis 9 (COP9) signalosome results in altered neddylation of cullin 4A and impairs lenalidomide-dependent CRL4CRBN activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4CRBN, with UBE2D3 acting to prime targets via monoubiquitination and UBE2G1 functioning to extend polyubiquitin chains with lysine 48 linkages. The validation of UBE2D3 and UBE2G1 highlights the functional capacity of CRISPR-Cas9 screening to identify E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase complex pairings. More broadly, these findings establish key proteins required for lenalidomide-dependent CRL4CRBN function in myeloma and inform potential mechanisms of drug resistance.

CK1α and IRF4 are essential and independent effectors of immunomodulatory drugs in primary effusion lymphoma

AbstractPrimary effusion lymphoma (PEL) is an aggressive cancer with few treatment options. The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been shown to kill PEL cell lines, and lenalidomide is in clinical trials against PEL. IMiDs bind to the CRL4CRBN E3 ubiquitin ligase complex, leading to the acquisition of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), casein kinase 1 α (CK1α), and zinc finger protein 91 (ZFP91) as neosubstrates. IMiDs are effective against multiple myeloma because of degradation of IKZF1 and IKZF3 and the consequent loss of interferon regulatory factor 4 (IRF4) and MYC expression. Lenalidomide is also effective in chromosome 5q deletion–associated myelodysplastic syndrome as a result of degradation of CK1α. An essential IKZF1-IRF4-MYC axis has recently been proposed to underlie the toxicity of IMiDs in PEL. Here, we further investigate IMiD effectors in PEL cell lines, based on genome-wide CRISPR/Cas9 screens for essential human genes. These screens and extensive validation experiments show that, of the 4 neosubstrates, only CK1α is essential for the survival of PEL cell lines. In contrast, IKZF1 and IKZF3 are dispensable, individually or in combination. IRF4 was critical in all 8 PEL cell lines tested, and surprisingly, IMiDs triggered downregulation of IRF4 expression independently of both IKZF1 and IKZF3. Reexpression of CK1α and/or IRF4 partially rescued PEL cell lines from IMiD-mediated toxicity. In conclusion, IMiD toxicity in PEL cell lines is independent of IKZF1 and IKZF3 but proceeds through degradation of the neosubstrate CK1α and downregulation of IRF4.

Surface expression of Cytokine Receptor-Like Factor 2 increases risk of relapse in pediatric acute lymphoblastic leukemia patients harboring IKZF1 deletions.

We prospectively examined whether surface expression of Cytokine Receptor-Like Factor 2 (CRLF2) on leukemic blasts is associated with survival and induction treatment response in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Flow cytometric analysis of bone marrow-derived leukemia cells revealed that 7.51% (29/286) of 386 pediatric BCP-ALL patients were CRLF2-positive (CRLF2pos) at diagnosis. The median minimal residual disease (MRD) was lower in CRLF2pos than CRLF2-negative (CRLF2neg) patients on day 15 (MRD15) after induction therapy [0.01% (0.001-0.42%) vs. 0.45% (0.05-3.50%); p=0.001]. By contrast, the MRD15 was higher in Ikaros family Zinc Finger Protein 1 (IKZF1)-deleted BCP-ALL patients than in BCP-ALL patients without IKZF1 deletions [1.18% (0.06-12.0%) vs 0.33% (0.03-2.6%); p=0.003]. Subgroup analysis showed that MRD15 levels were lower in IKZF1Δ/CRLF2pos patients than in IKZF1Δ/CRLF2neg patients [0.1% (0.02-5.06%) vs. 2.9% (0.25-12%); p=0.005]. Furthermore, MRD15 levels were higher in IKZF1WT/CRLF2neg patients than in IKZF1WT/CRLF2pos patients [0.40% (0.04-2.7%) vs. 0.001% (0.001-0.01%)]. Despite the low MRD15 levels, IKZF1Δ/CRLF2pos patients showed poorer relapse-free survival (RFS) than other patient groups (p=0.003). These findings demonstrate that surface CRLF2 expression is associated with increased risk of relapse in pediatric BCP-ALL patients harboring IKZF1 deletions.

P97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.

Lenalidomide Effectively Induces Philadelphia-Chromosome Positive ALL Cells into Apoptosis in Synergy with Tyrosine Kinase Inhibitor

[Background] Lenalidomide (LMD) has been effectively used for the treatment of hematologic malignancies such as multiple myeloma and 5q-MDS. Recently, LMD was shown to bind the celebron (CRBN) E3 ubiquitin ligase complex and degrade Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) via ubiquitin-proteasome system in myeloma cells, which leads to decrease in expression of downstream targets such as IRF4 and c-Myc. The IKZF1 is an essential transcription factor for the development of all lymphoid lineages particularly in the development of B- cell progenitors (BCP), and its gene consists of 8 exons including exons 4-6 and exon 8 encoding DNA-binding and dimerization domains, respectively. Genomic heterozygous deletions in IKZF1 are found in ~15% childhood BCP-ALL and the most frequent deletions affect the whole gene or exons 4-7 resulting in haploinsufficiency or expression of a dominant negative isoform. The IKZF1 deletions are now a hallmark for refractory nature of BCP-ALL linking to an unfavorable clinical outcome, and the frequency of IKZF1deletion is remarkably high (~70%) in Philadelphia-chromosome positive (Ph+) ALL cases. It is thus of great interest to examine LMD's effects on Ph+ALL cells.[Materials mad Methods] A total of 7 Ph+ALL cell lines with IKZF1 deletions (4 in exons 4-7, 2 in exons 2-7, and 1 in exons 1-8) were used to examine LMD's effects. The synergic effects of LMD with tyrosine kinase inhibitor (TKI; imatinib, dasatinib) were also examined. The ex vivo studies using NOD/Shi-scid, IL-2Rgnull (NOG) mice model were performed.[Results] 1. We first performed [3H]-thymidine uptake assays in the presence or absence of 20µM LMD for 3-6 days, and found that thymidine uptakes were variably inhibited (8~65%) after LMD treatment and two of sensitive cell lines showed a marked inhibition in a dose and a time dependent manners of LMD (~75%). Of importance, their thymidine uputakes were further inhibited (~95%) by LMD in the presence of the imatinib in a dose dependent manner (0.05-0.5nM). 2. To access the inhibition of thymidine uptake, we performed alamarBlue cytotoxic assays using the sensiitive cell line KOPN57bi, and found that the cell numbers modestly decreased by LMD (20 µM) or imatinib (0.5nM) , and markedly decreased (up to 35% at day 4) by LMD+imatinib. In flow cytometric analysis, KOPN57bi treated with LMD or imatinib showed cell cycle arrest alone, while the cell line treated with LMD+imatinib showed a marked increase in the subdiploid (42.3%), Annexin V-positive (72.0%), and active caspase 3-positive (31.8%) populations at day 4 after trreatment. The pancaspase inhibitor Z-VAD-FMK completely inhibited induction of cell death (apoptosis) by LMD+imatinib. These results were also obtained by use of another TKI dasatinib instead of imatinib. 3. We examined changes in expression of IKZF1 on Western blot using anti-C-terminal IKZF1 antibody in KOPN57bi (deleted in exons 4-7) 24h after LMD and/or TKI treatment, and found that several alternative splicing forms translated from intact IKZF1 disappear by LMD, but expression of a dominant negative Ik6 isoform was not affected by LMD presumably due to a lack of the CRBN binding site (residue Gln 146:exon5) in Ik6 isoform. IRF4 was also down-regulated by LMD and further in the presence of TKI. 4. We performed ex vivo studies using NOG mice. After intravenous injection of KOPN57bi (2x106cells), mice were orally administrated with saline (control, n=4), LMD (30mg/kg/d, n=5), imatinib (150mg/kg/d, n=5) , or LMD+imatinib (n=4) for 10 days (day 0,1,2,3,4, 7,8, 9,10,and 11) and percentages of human CD45 positive population in bone marrow mononuclear cells were examined at day 14. Human CD45+ cells showed 29.7±4.3% (mean±SEM) and 30.3±1.5% in control and imatinib-treated mice, respectively, whereas they were significantly (p=0.0002) decreased to 2.1±0.4% in LMD-treated mice. Of importance, they were further decreased to 0.3±0.4% in LMD+imatinib treated mice (p=0.0051), suggesting that combined treatment of LMD and imatinib should be very effective in xenograft mice models.[Conclusion] LMD renderd Ph+ALL cells a marked cell cycle arrest by completely abrogating the remaining IKZF1 function, and effectively induced them into apoptosis in synergy with TKI. Orally administration of LMD and TKI might become a high QOL treatment option for the treatment of Ph+ALL patients particularly for those with a higher age and organ dysfunctions. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5445: Dual-color immunohistochemistry assays for measuring Aiolos and Ikaros proteins in multiple myeloma patient samples

Abstract IMiDs® immunomodulatory drugs Lenalidomide (LEN) and Pomalidomide (POM) are antineoplastic agents that have a significant clinical impact in the treatment of patients with multiple myeloma (MM). Recent studies indicate that the anti-proliferative activity of LEN and POM in MM cells is associated with ubiquitination and degradation of the Ikaros family zinc finger protein transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1), the substrates of the Cereblon (CRBN)-dependent Cullin 4 E3-ligase complex. Measurement of Aiolos and Ikaros protein levels in MM patient samples is critical for future correlative studies. Here we describe the development and validation of dual IHC Aiolos/CD138 and Ikaros/CD138 assays using the highly specific Aiolos rabbit monoclonal antibody CGN-9-9-7, and a commercial Ikaros rabbit polyclonal antibody H-100 in combination with a commercial CD138 antibody. Aiolos and Ikaros immunoreactivity in CD138+ MM cells is scored using the semi-quantitative H-score method. H-scores range from 0 to 300 and are the sum of the products of the percentage of tumor cells (0-100%) and the intensity of staining (0-3). The assays were specific and detected high Aiolos and Ikaros expression in the positive control OCI-Ly10 lymphoma cell line, and low Aiolos and Ikaros levels in POM treated OCI-Ly10 cells. These results were confirmed via Western Blot analysis of Aiolos and Ikaros expression data in these cells lines. Assay precision was examined via staining serial sections of the same MM tumor across three different days and deemed acceptable. The dual IHC assays were used to evaluate bone marrow core biopsies or aspirate clots from 22 MM patients and H-scores were determined by a board-certified pathologist. The dual IHC assays detected a wide range of Aiolos or Ikaros immunoreactivity in the 22 MM cases, with nuclear H-scores ranging from 10 to 260 for Aiolos and 40 to 240 for Ikaros. In conclusion, the dual Aiolos/CD138 and Ikaros/CD138 IHC assays were developed to provide a specific and reliable semi-quantitative method to evaluate Aiolos and Ikaros protein levels in both bone marrow core biopsies and clots from MM patients. These assays are currently being used in clinical trials to assess the importance of Aiolos and Ikaros as biomarkers in MM patients treated with IMiDs® immunomodulatory drugs. Citation Format: Yan Ren, Maria Wang, Suzana Couto, Donna Hansel, Anita K. Gandhi, Patrick Hagner, Anjan Thakurta, Rajesh Chopra, Mike Breider. Dual-color immunohistochemistry assays for measuring Aiolos and Ikaros proteins in multiple myeloma patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2015-5445

Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21WAF1/Cip1-dependent mechanism independent of functional p53

AbstractLenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21WAF1/Cip1 (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.

IKZF1 deletion is associated with a poor outcome in pediatric B‐cell precursor acute lymphoblastic leukemia in Japan

Genetic alterations of Ikaros family zinc finger protein 1 (IKZF1), point mutations in Janus kinase 2 (JAK2), and overexpression of cytokine receptor-like factor 2 (CRLF2) were recently reported to be associated with poor outcomes in pediatric B-cell precursor (BCP)-ALL. Herein, we conducted genetic analyses of IKZF1 deletion, point mutation of JAK2 exon 16, 17, and 21, CRLF2 expression, the presence of P2RY8-CRLF2 fusion and F232C mutation in CRLF2 in 202 pediatric BCP-ALL patients newly diagnosed and registered in Japan Childhood Leukemia Study ALL02 protocol to find out if alterations in these genes are determinants of poor outcome. All patients showed good response to initial prednisolone (PSL) treatment. Ph+, infantile, and Down syndrome–associated ALL were excluded. Deletion of IKZF1 occurred in 19/202 patients (9.4%) and CRLF2 overexpression occurred in 16/107 (15.0%), which are similar to previous reports. Patients with IKZF1 deletion had reduced event-free survival (EFS) and overall survival (OS) compared to those in patients without IKZF1 deletion (5-year EFS, 62.7% vs. 88.8%, 5-year OS, 71.8% vs. 90.2%). Our data also showed significantly inferior 5-year EFS (48.6% vs. 84.7%, log rank P = 0.0003) and 5-year OS (62.3% vs. 85.4%, log rank P = 0.009) in NCI-HR patients (n = 97). JAK2 mutations and P2RY8-CRLF2 fusion were rarely detected. IKZF1 deletion was identified as adverse prognostic factor even in pediatric BCP-ALL in NCI-HR showing good response to PSL.