A review on the treatment of multiple myeloma with small molecular agents in the past five years.

Multiple myeloma is currently incurable, and the incidence rate is increasing year by year worldwide. Although in recent years the combined treatment plan based on proteasome inhibitors and immunomodulatory drugs has greatly improved the treatment effect of multiple myeloma, most patients still relapse and become resistant to current treatments. To solve this problem, scientists are committed to developing drugs with higher specificity, such as iberdomide, which is highly specific to ikaros and aiolos. This review aims to focus on the small molecular agents that are being researched/clinically used for the treatment of multiple myeloma, including the target mechanism, structure-activity relationship and application prospects of small molecular agents.

Germline IKAROS dimerization haploinsufficiency causes hematologic cytopenias and malignancies.

IKAROS is a transcription factor forming homo- and heterodimers and regulating lymphocyte development and function. Germline mutations affecting the IKAROS N-terminal DNA binding domain, acting in a haploinsufficient or dominant-negative manner, cause immunodeficiency. Herein, we describe 4 germline heterozygous IKAROS variants affecting its C-terminal dimerization domain, via haploinsufficiency, in 4 unrelated families. Index patients presented with hematologic disease consisting of cytopenias (thrombocytopenia, anemia, neutropenia)/Evans syndrome and malignancies (T-cell acute lymphoblastic leukemia, Burkitt lymphoma). These dimerization defective mutants disrupt homo- and heterodimerization in a complete or partial manner, but they do not affect the wild-type allele function. Moreover, they alter key mechanisms of IKAROS gene regulation, including sumoylation, protein stability, and the recruitment of the nucleosome remodeling and deacetylase complex; none affected in N-terminal DNA binding defects. These C-terminal dimerization mutations are largely associated with hematologic disorders, display dimerization haploinsufficiency and incomplete clinical penetrance, and differ from previously reported allelic variants in their mechanism of action. Dimerization mutants contribute to the growing spectrum of IKAROS-associated diseases displaying a genotype-phenotype correlation.

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

Dynamic Imaging of Small Molecule Induced Protein-Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach.

Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein-protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.

Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency.

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

Protein Degradation via CRL4CRBN Ubiquitin Ligase: Discovery and Structure-Activity Relationships of Novel Glutarimide Analogs That Promote Degradation of Aiolos and/or GSPT1.

We previously disclosed the identification of cereblon modulator 3 (CC-885), with potent antitumor activity mediated through the degradation of GSPT1. We describe herein the structure-activity relationships for analogs of 3 with exploration of the structurally related dioxoisoindoline class. The observed activity of protein degradation could in part be rationalized through docking into the previously disclosed 3-CRBN-GSPT1 cocrystal ternary complex. For SAR that could not be rationalized through the cocrystal complex, we sought to predict SAR through a QSAR model developed in house. Through these analyses, selective protein degradation could be achieved between the two proteins of interest, GSPT1 and Aiolos.

Ikaros: Exploiting and targeting the hematopoietic stem cell niche in B-progenitor acute lymphoblastic leukemia.

Genetic alterations of IKZF1 encoding the lymphoid transcription factor IKAROS are a hallmark of high-risk B-progenitor acute lymphoblastic leukemia (ALL), such as BCR-ABL1-positive (Ph+) and Ph-like ALL, and are associated with poor outcome even in the era of contemporary chemotherapy incorporating tyrosine kinase inhibitors. Recent experimental mouse modeling of B-progenitor ALL has shown that IKZF1 alterations have multiple effects, including arresting differentiation, skewing lineage of leukemia from myeloid to lymphoid, and, in Ph+ leukemia, conferring resistance to tyrosine kinase inhibitor (TKI) therapy without abrogating ABL1 inhibition. These effects are in part mediated by acquisition of an aberrant hematopoietic stem cell-like program accompanied by induction of cell surface expression of stem cell and adhesion molecules that mediate extravascular invasion and residence in the niche and activation of integrin signaling pathways. These effects can be exploited therapeutically using several approaches. IKZF1 alterations also result in upregulation of RXRA that encodes part of the heterodimeric retinoic acid X receptor. Rexinoids, a synthetic class of retinoids that bind specifically to retinoid "X" receptors such as bexarotene potently reverse aberrant adhesion and niche mislocalization in vivo and induce differentiation and cell cycle arrest. Focal adhesion kinase inhibitors block the downstream integrin-mediated signaling, reverse adhesion, and niche mislocalization. Both agents act synergistically with TKIs to prolong survival of Ph+ ALL in mouse and human xenograft model, with long-term remission induced by focal adhesion kinase inhibitors. Therefore, these findings provide important new conceptual insights into the mechanisms by which IKZF1 alterations result in drug resistance and indicate that therapeutic strategies directed against the pathways deregulated by mutation, rather than attempting to restore IKZF1 expression directly, represent promising therapeutic approaches in this disease.

A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.

Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase.

Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4(CRBN)) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4(CRBN) substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clinical efficacy in the treatment of multiple myeloma and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS), respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α ß-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4(CRBN) is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.

Protein signaling and regulation of gene transcription in leukemia: role of the Casein Kinase II-Ikaros axis.

Protein signaling and regulation of gene expression are the two major mechanisms that regulate cellular proliferation in leukemia. Discerning the function of these processes is essential for understanding the pathogenesis of leukemia and for developing the targeted therapies. Here, we provide an overview of one of the mechanisms that regulates gene transcription in leukemia. This mechanism involves the direct interaction between Casein Kinase II (CK2) and the Ikaros transcription factor. Ikaros (IKZF1) functions as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Impaired Ikaros function results in the development of high-risk leukemia. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. In vivo, Ikaros is a target for CK2, a pro-oncogenic kinase. CK2 directly phosphorylates Ikaros at multiple amino acids. Functional experiments showed that CK2-mediated phosphorylation of Ikaros, regulates Ikaros' DNA binding affinity, subcellular localization and protein stability. Recent studies revealed that phosphorylation of Ikaros by CK2 regulates Ikaros binding and repression of the terminal deoxytransferase (TdT) gene in normal thymocytes and in T-cell ALL. Available data suggest that the oncogenic activity of CK2 in leukemia involves functional inactivation of Ikaros and provide a rationale for CK2 inhibitors as a potential treatment for ALL.

[Thalidomide, cereblon and multiple myeloma].

Cereblon was identified as a direct target of thalidomide by Prof. H. Handa, and this pioneering work triggered active research on IMiDs (immunomodulatory drugs), which include thalidomide-derivatives, such as lenalidomide and pomalidomide. These small molecules have been shown to bind to cereblon (CRBN) to modulate its activity as a substrate receptor. In addition, structural analyses on CRBN have revealed unique actions of these small agents, by which degradation of transcription factors is controlled in a specific and unique way. I summarize recent progress on CRBN-CRLA ubiquitin ligase and IMiDs, focusing on the therapeutic application of these drugs for treatment of multiple myeloma.

[C2H2 zinc-finger recognition of biomolecules].

C2H2 zinc-finger motif presents in 3% of proteins that are encoded in the human genome, and has the abilities to recognize DNA, RNA and protein. With nearly 3 decades of efforts, the mechanisms of zinc-finger mediated biomolecule recognitions have been studied to various extents. Zinc-finger binds into the major groove of DNA double helix, establishes an one-to-one recognition format between DNA bases and certain amino acids in a zinc-finger, and achieves specificity based on DNA sequences. While RNA molecules show a large variety in their structures, zinc-finger recognizes RNA through the collected information of specially displayed bases and special backbone folding. Initial studies have been performed on zinc-finger mediated protein-protein interactions. Existing data indicate multiple recognition modes. The studies on molecular mechanism have supported the development of engineered zinc-fingers, which have been introduced into applications. For its wide existence, large functional diversity and potential in translational applications, zinc-finger deserves a systematic study in every aspect.

A novel, non-canonical splice variant of the Ikaros gene is aberrantly expressed in B-cell lymphoproliferative disorders.

The Ikaros gene encodes a Krüppel-like zinc-finger transcription factor involved in hematopoiesis regulation. Ikaros has been established as one of the most clinically relevant tumor suppressors in several hematological malignancies. In fact, expression of dominant negative Ikaros isoforms is associated with adult B-cell acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia and adult and juvenile chronic myeloid leukemia. Here, we report the isolation of a novel, non-canonical Ikaros splice variant, called Ikaros 11 (Ik11). Ik11 is structurally related to known dominant negative Ikaros isoforms, due to the lack of a functional DNA-binding domain. Interestingly, Ik11 is the first Ikaros splice variant missing the transcriptional activation domain. Indeed, we demonstrated that Ik11 works as a dominant negative protein, being able to dimerize with Ikaros DNA-binding isoforms and inhibit their functions, at least in part by retaining them in the cytoplasm. Notably, we demonstrated that Ik11 is the first dominant negative Ikaros isoform to be aberrantly expressed in B-cell lymphoproliferative disorders, such as chronic lymphocytic leukemia. Aberrant expression of Ik11 interferes with both proliferation and apoptotic pathways, providing a mechanism for Ik11 involvement in tumor pathogenesis. Thus, Ik11 could represent a novel marker for B-cell lymphoproliferative disorders.

Direct protein interactions are responsible for Ikaros-GATA and Ikaros-Cdk9 cooperativeness in hematopoietic cells.

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

Pegasus, the 'atypical' Ikaros family member, influences left-right asymmetry and regulates pitx2 expression.

Members of the Ikaros family of zinc-finger transcription factors have been shown to be critical for immune and blood cell development. However, the role of the most divergent family member, Pegasus, has remained elusive, although it shows conservation to invertebrate Hunchback proteins that influence embryonic patterning through regulation of homeodomain genes. Zebrafish was employed as a relevant model to investigate the function of Pegasus since it possesses a single pegasus orthologue with high homology to its mammalian counterparts. During zebrafish embryogenesis pegasus transcripts were initially maternally-derived and later replaced by zygotic expression in the diencephalon, tectum, hindbrain, thymus, eye, and ultimately the exocrine pancreas and intestine. Morpholino-mediated knockdown of the zebrafish pegasus gene resulted in disrupted left-right asymmetry of the gut and pancreas. Molecular analysis indicated that zebrafish Pegasus localised to the nucleus in discrete non-nucleolar structures and bound the 'atypical' DNA sequence GN3GN2G, confirming its presumed role as a transcriptional regulator. In vivo transcriptome analysis identified candidate target genes, several of which encoded homeodomain transcription factors. One of these, pitx2, implicated in left-right asymmetry, possessed appropriate 'atypical' Pegasus binding sites in its promoter. Knockdown of Pegasus affected both the level and asymmetry of pitx2 expression, as well as disrupting the asymmetry of the lefty2 and spaw genes, explaining the perturbed left-right patterning in pegasus morphants. Collectively these results provide the first definitive insights into the in vivo role of Pegasus, supporting the notion that it acts as a broader regulator of development, with potential parallels to the related invertebrate Hunchback proteins.

Ikaros and tumor suppression in acute lymphoblastic leukemia.

The Ikzf1 gene encodes Ikaros-a DNA-binding zinc finger protein. Ikaros functions as a regulator of gene expression and chromatin remodeling. The biological roles of Ikaros include regulating the development and function of the immune system and acting as a master regulator of hematopoietic differentiation. Genomic profiling studies identified Ikzf1 as an important tumor suppressor in acute lymphoblastic leukemia (ALL), particularly in ALL that is associated with poor prognosis. This review summarizes currently available data regarding the structure and function of Ikaros, the clinical relevance of genetic inactivation of Ikzf1, and signal transduction pathways that regulate Ikaros function.

Evolution of the Ikaros gene family: implications for the origins of adaptive immunity.

Members of the Ikaros family of transcription factors are important for immune system development. Analysis of Ikaros-related genes from a range of species suggests the Ikaros family derived from a primordial gene, possibly related to the present-day protostome Hunchback genes. This duplicated before the divergence of urochordates to produce two distinct lineages: one that generated the Ikaros factor-like (IFL) 2 genes of urochordates/lower vertebrates and the Pegasus genes of higher vertebrates, and one that generated the IFL1 genes of urochordates/lower vertebrates, the IKFL1 and IKFL2 genes of agnathans and the remaining four Ikaros members of higher vertebrates. Expansion of the IFL1 lineage most likely occurred via the two intervening rounds of whole genome duplication. A proposed third whole genome duplication in teleost fish produced a further increase in complexity of the gene family with additional Pegasus and Eos members. These findings question the use of IFL sequences as evidence for the existence of adaptive immunity in early chordates and vertebrates. Instead, this study is consistent with a later emergence of adaptive immunity coincident with the appearance of the definitive lymphoid markers Ikaros, Aiolos, and Helios.

BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros.

The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL.

Recruitment of ikaros to pericentromeric heterochromatin is regulated by phosphorylation.

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.

Ikaros is expressed in developing striatal neurons and involved in enkephalinergic differentiation.

The Ikaros (Ik) gene encodes alternatively spliced zinc-finger proteins originally identified in developing hematopoietic organs and acts as master regulator of lymphoid development. During our search for transcription factors that control the developmental expression of the enkephalin (ENK) gene we found that Ik-1 and Ik-2 isoforms are specifically expressed in the embryonic striatum and bind the Ik-like cis-regulatory DNA element present on the ENK gene. Ik proteins are expressed by both proliferating (BrdU+/nestin+) and by post-mitotic differentiating (MAP2+) cells in the developing striatum between embryonic day 12 and post-natal day 2 and mRNAs encoding for the Ik and ENK genes are co-expressed by a subset of differentiating striatal neurons. Blocking the DNA binding of Ik proteins in differentiating embryonic striatal neuronal cultures resulted in decreased ENK expression and mutant animals lacking the DNA-binding domain of Ik had a deficit in the number of ENK but not in dynorphin or substance P mRNA+ cells. Animals lacking the protein interaction domain of Ik showed no deficit. These results demonstrate that Ik-1 and Ik-2 proteins through their DNA binding act as positive regulators of ENK gene expression in the developing striatum and participate in regulating enkephalinergic differentiation.