AI-10-49
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AI-10-49 is a small molecule inhibitor of leukemic
oncoprotein An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels.
CBFβ-SMHHC developed by the laboratory of John Bushweller (
University of Virginia The University of Virginia (UVA) is a Public university#United States, public research university in Charlottesville, Virginia, United States. It was founded in 1819 by Thomas Jefferson and contains his The Lawn, Academical Village, a World H ...
) with efficacy demonstrated by the laboratories of Lucio H. Castilla (
University of Massachusetts Medical School The UMass Chan Medical School is a public medical school in Worcester, Massachusetts. It is part of the University of Massachusetts system. It consists of three schools: the T.H. Chan School of Medicine, the Morningside Graduate School of Biom ...
) and Monica Guzman (
Cornell University Cornell University is a Private university, private Ivy League research university based in Ithaca, New York, United States. The university was co-founded by American philanthropist Ezra Cornell and historian and educator Andrew Dickson W ...
). AI-10-49
allosterically In the fields of biochemistry and pharmacology an allosteric regulator (or allosteric modulator) is a substance that binds to a site on an enzyme or receptor distinct from the active site, resulting in a conformational change that alters the p ...
binds to CBFβ-SMMHC and disrupts protein-protein interaction between CBFβ-SMMHC and tumor suppressor
RUNX1 Runt-related transcription factor 1 (RUNX1) also known as acute myeloid leukemia 1 protein (AML1) or core-binding factor subunit alpha-2 (CBFA2) and it is a protein that is encoded by the ''RUNX1'' gene, in humans. RUNX1 is a transcription facto ...
. This inhibitor is under development as an anti-leukemic drug.


Core binding factors

Core-binding factor (CBF) is a
heterodimeric In biochemistry, a protein dimer is a macromolecular complex or multimer formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ...
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
composed by the CBFβ and RUNX subunits (the latter is encoded by RUNX1, RUNX2, or RUNX3 genes). CBF plays critical roles in most hematopoietic lineages, regulating gene expression of a variety of genes associated with
cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...
, differentiation, signaling and
adhesion Adhesion is the tendency of dissimilar particles or interface (matter), surfaces to cling to one another. (Cohesion (chemistry), Cohesion refers to the tendency of similar or identical particles and surfaces to cling to one another.) The ...
. In hematopoiesis, CBF regulates
progenitor cell A progenitor cell is a biological cell that can differentiate into a specific cell type. Stem cells and progenitor cells have this ability in common. However, stem cells are less specified than progenitor cells. Progenitor cells can only diffe ...
fate decisions and differentiation at multiple levels. The function of CBF is essential for the emergence of embryonic hematopoietic stem cells (HSCs) and establishment of definitive
hematopoiesis Haematopoiesis (; ; also hematopoiesis in American English, sometimes h(a)emopoiesis) is the formation of blood cellular components. All cellular blood components are derived from haematopoietic stem cells. In a healthy adult human, roughly ten ...
at midgestation. Similarly, in adult hematopoiesis, CBF regulates the frequency and differentiation of HSCs,
lymphoid The lymphatic system, or lymphoid system, is an organ system in vertebrates that is part of the immune system and complementary to the circulatory system. It consists of a large network of lymphatic vessels, lymph nodes, lymphoid organs, lympha ...
and
myeloid Myeloid tissue, in the bone marrow sense of the word '' myeloid'' ('' myelo-'' + '' -oid''), is tissue of bone marrow, of bone marrow cell lineage, or resembling bone marrow, and myelogenous tissue (''myelo-'' + '' -genous'') is any tissue ...
progenitors, establishing CBF as a master regulator of hematopoietic homeostasis.


Core binding factors and leukemia

CBF members are frequent targets of mutations and rearrangements in human
leukemia Leukemia ( also spelled leukaemia; pronounced ) is a group of blood cancers that usually begin in the bone marrow and produce high numbers of abnormal blood cells. These blood cells are not fully developed and are called ''blasts'' or '' ...
. Point-mutations in RUNX1 gene have been reported in patients with familial platelet disorder, myeloid dysplastic syndrome, and chronic myelomonocytic leukemia. In addition, RUNX1 mutations have also been reported in Acute
myeloid leukemia Myeloid leukemia is a type of leukemia affecting myeloid tissue. Types include: * Acute myeloid leukemia: A cancer of the myeloid line of blood cells, characterized by the rapid growth of myeloblasts that build up in the bone marrow and blood and ...
(AML). The RUNX1 and CBFB genes are targets of chromosome rearrangements that create
oncogenic Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...
fusion genes in leukemia. The
chromosome translocation In genetics, chromosome translocation is a phenomenon that results in unusual rearrangement of chromosomes. This includes "balanced" and "unbalanced" translocation, with three main types: "reciprocal", "nonreciprocal" and "Robertsonian" transloc ...
t(12;21) (p13.1;q22) causes the fusion of the ETS variant 6 (''
ETV6 ETV6 (i.e. translocation-Ets-leukemia virus) protein is a transcription factor that in humans is encoded by the ''ETV6'' (previously known as ''TEL'') gene. The ETV6 protein regulates the development and growth of diverse cell types, particularly ...
'') and RUNX1 genes results in ''ETV6-RUNX1'' gene fusion and is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL). The "core binding factor AML" (CBF AML) HO classificationis the most common group of AML, including groups with the chromosome rearrangements inv(16)(p13q22) and t(8;21)(q22;q22). The chromosome translocation t(8;21)(q22;q22) creates the RUNX1-ETO fusion gene, which is expressed in FAB subtype M2 AML samples. The pericentric
chromosome inversion An inversion is a chromosome rearrangement in which a segment of a chromosome becomes inverted within its original position. An inversion occurs when a chromosome undergoes a two breaks within the chromosomal arm, and the segment between the two b ...
inv(16)(p13q22) creates the CBFB-MYH11 fusion gene, which encodes the CBFβ-SMMHC fusion protein.


Inv(16) leukemia

The inv(16) is present in all M4Eo subtype AML, representing one of the most common change in AML, and accounting for ≈12% of de novo human AML. Studies by various laboratories have established that CBFβ-SMMHC acts as a dominant repressor of CBF function in vivo and specifically blocks lymphoid and myeloid lineage differentiation. Treatment of AML varies based on the prognosis and mutations identified in the patient sample. Current treatment for inv(16) AML uses
chemotherapy Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...
drugs, such as
doxorubicin Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used toge ...
and
cytarabine Cytarabine, also known as cytosine arabinoside (ara-C), is a chemotherapy medication used to treat acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and non-Hodgkin's lymphoma. It is given b ...
, with an estimated 5-year overall survival of 60% in young patients and only 20% in the elderly.


Discovery

CBFβ-SMMHC outcompetes CBFβ for binding to RUNX1 by direct protein-protein interaction. Using a fluorescence resonance energy transfer (FRET) based assay, AI-4-57 was discovered as the lead compound which can inhibit CBFβ-SMHHC –RUNX1 protein-protein interaction. To get good in vivo
pharmacokinetic Pharmacokinetics (from Ancient Greek ''pharmakon'' "drug" and ''kinetikos'' "moving, putting in motion"; see chemical kinetics), sometimes abbreviated as PK, is a branch of pharmacology dedicated to describing how the body affects a specific subs ...
s,
selectivity Selectivity may refer to: Psychology and behaviour * Choice, making a selection among options * Discrimination, the ability to recognize differences * Socioemotional selectivity theory, in social psychology Engineering * Selectivity (radio), a ...
and potency AI-10-49 was developed which has a seven atom
polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular wei ...
-based linker and a trifluoromethoxy substitution. This molecule releases RUNX1 from CBFβ-SMHHC specifically and restores the RUNX1 transcriptional program in inv(16) positive human leukemic cells. Viability assays showed AI-10-49 has an
IC50 Half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e. ...
of 0.6μM. Pharmacokinetic studies showed that AI-10-49 has half-life of 380 minutes in mouse plasma. AI-10-49 prolonged the survival of mice transplanted with CBFβ-SMHHC leukemic cells without any signs of toxicity. AI-10-49 reduced viability and colony forming ability of human primary inv(16) leukemic blast cells, without affecting normal human bone marrow cells as wells non- inv(16) primary human leukemic blast cells. Overall, these findings validate inhibition of RUNX1- CBFβ-SMMHC protein-protein interaction as a novel therapeutic avenue for leukemia with inv(16) and AI-10-49 as a specific inhibitor of CBFβ-SMHHC oncoprotein. The discovery of AI-10-49 provides additional evidence that transcription factor drivers of cancer can be directly targeted. AI-10-49 belongs to a select group of protein-protein interaction inhibitors that has been shown to have specific and potent efficiency without toxicity in cancer therapy.


Mechanism of action of AI-10-49

The mechanism of action of AI-10-49 was recently elucidated by the Castilla laboratory at
University of Massachusetts Medical School The UMass Chan Medical School is a public medical school in Worcester, Massachusetts. It is part of the University of Massachusetts system. It consists of three schools: the T.H. Chan School of Medicine, the Morningside Graduate School of Biom ...
. Gene Set Enrichment Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 identified the deregulation of a MYC signature, including cell cycle, ribosome biogenesis and metabolism. MYC shRNA knockdown induced apoptosis of inv(16) AML cells, and MYC overexpression partially rescued AI-10-49 induced apoptosis. Furthermore, mouse leukemia cells transduced with Myc shRNAs showed significant delay in leukemic latency upon transplantation, validating the requirement of MYC in inv(16) AML maintenance in vivo. Pharmacologic inhibition of MYC activity, using a combined treatment with AI-10-49 and the BET-family bromodomain inhibitor JQ1, revealed a strong synergy in inv(16) AML cells and a significant delay in leukemia latency in mice. ChIP-seq and ATAC-seq analysis revealed that inhibition of the CBFβ-SMHHC–RUNX1 protein–protein interaction by AI-10-49 results in increased RUNX1 occupancy at three MYC distal enhancers downstream from MYC transcription start site. Deletion of the RUNX1 binding site in these enhancers by genome editing (CRISPR/Cas9) reduced MYC transcript levels and the viability of inv(16) AML cells, indicating that each one of these enhancers plays a critical role in regulating MYC levels and sustaining the survival of inv(16) AML cells. Analysis of enhancer-promoter interactions by chromosome conformation capture carbon copy (5C) in inv(16) AML cells revealed that the three enhancers are physically connected with each other and to the MYC promoter. Analysis of chromatin immunoprecipitation revealed that AI-10-49 treatment results in the displacement of the SWI/SNF complex component BRG1 and RUNX1 mediated recruitment of polycomb-repressive complex 1 (PRC1) component RING1B at the three MYC enhancers. Taken together, these results demonstrate that AI-10-49 treatment induces an acute release of RUNX1, increases RUNX1 occupancy at MYC enhancers, and disrupts enhancer chromatin dynamics which in turn induces apoptosis by repressing MYC. Furthermore, this study suggests that combined treatment of inv(16) AML with AI-10-49 and BET-family inhibitors may represent a promising targeted therapy.


Protein-protein interaction inhibitors in cancer therapy

Targeting protein-protein interaction with small molecule is known to be extremely difficult due to the fact that binding regions consist of wide, shallow surfaces. There are few protein-protein interaction inhibitors with specific and non-toxic effect in various cancer types. The first and best characterized protein-protein interaction inhibitor in cancer therapy is
Nutlin Nutlins are a family of small molecule, ''cis''-imidazoline analogs, which inhibit the interaction between MDM2 and tumor suppressor p53, stabilizing p53 and triggering cell death and senescence. Three nutlins were discovered in the initial sma ...
. Nutlin inhibits the interaction between HDM2 and tumour suppressor
p53 p53, also known as tumor protein p53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory transcription factor protein that is often mutated in human cancers. The p53 proteins (originally thou ...
. After the discovery of Nutlin, more than 20 small molecule inhibitors have been developed by academic institutes and pharmaceutical companies of which 8 inhibitors are under Phase 1
clinical trials Clinical trials are prospective biomedical or behavioral research studies on human subject research, human participants designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel v ...
. Other examples for protein-protein interaction inhibitors include JQ1 (inhibits the interaction between acetylated histones and
BRD4 Bromodomain-containing protein 4 is a protein that in humans is encoded by the ''BRD4'' gene. BRD4 is a member of the BET (bromodomain and extra terminal domain) family, which also includes BRD2, BRD3, and BRDT. BRD4, similar to other BET fam ...
); 79-6 (inhibits
BCL6 Bcl-6 (B-cell lymphoma 6) is a protein that in humans is encoded by the ''BCL6'' gene. BCL6 is a master transcription factor for regulation of T follicular helper cells (TFH cells) proliferation. BCL6 has three evolutionary conserved structural d ...
BTB domain dimerization); MI-463 and MI-503 (inhibit Menin-MLL interaction) and ABT-737 (inhibits BCL2L1-BCL2 interaction).


References

{{Reflist Benzimidazoles Disubstituted pyridines Experimental cancer drugs Trifluoromethyl ethers