Cereblon; CC-90009 is a first-in-class cereblon E3 ligase modulating drug (CELMoD) that selectively targets GSPT1 (G1 to S phase transition 1, also known as eRF3a) for proteasomal degradation. It co-opts the CRL4CRBN E3 ubiquitin ligase complex to promote ubiquitination and degradation of GSPT1. [1]
In cellular degradation assays, CC-90009 degrades GSPT1 with an EC50 of 9 nM (at 4 hours) and a Dmax of 51%; at 20 hours, the EC50 remains 9 nM with a Dmax of 88%. It shows high selectivity for GSPT1 over other known neosubstrates such as IKZF1 and IKZF3 (EC50 > 10 μM). [2]

CC-90009 demonstrates potent antiproliferative activity in 10 out of 11 human AML cell lines tested, with much less effect on peripheral blood mononuclear cells from healthy donors and THLE-2 hepatocyte cells. [1]
In primary AML patient bone marrow samples, CC-90009 rapidly induces loss of viable leukemic cells. In 8 out of 9 patient samples, significant cell death was observed at 48 hours post-treatment, with nearly all leukemic cells eliminated within 96 hours. The growth inhibition of leukemic cells was 2- to 5-fold greater than that of normal lymphocytes from the same patients. In 9 of the first 10 patient samples examined, the average EC50 was approximately 6 nM. [1][2]
Mass spectrometry proteomics analysis in KG-1 cells showed that CC-90009 treatment (100 nM, 4 hours) selectively reduces GSPT1 protein abundance with minimal effects on the rest of the proteome. Immunoblotting confirmed specific degradation of GSPT1 without affecting other known CC-885 neosubstrates including IKZF1, HBS1L, and CK1α. [1]
CC-90009-induced GSPT1 degradation is blocked by proteasomal inhibition with bortezomib, by neddylation inhibition with MLN4924, or by CRBN knockout, confirming the CRL4CRBN-dependent mechanism. [1][2]
The degradation of GSPT1 by CC-90009 leads to activation of the integrated stress response pathway, including phosphorylation of eIF2α, accumulation of ATF4 and its transcriptional targets CHOP, ATF3, and DDIT4, and subsequent induction of apoptosis as evidenced by caspase-3 cleavage. [1][2]
In a cellular degradation assay, CC-90009 showed potent GSPT1 degradation with EC50 = 9 nM and Dmax = 51% at 4 hours; at 20 hours, the degradation depth increased to Dmax = 88% with the same EC50. It demonstrated >1000-fold selectivity for GSPT1 over IKZF1/3 (EC50 > 10 μM). [2]
Proteomic experiments with concentrations ranging from 0.20 μM to 3 μM and incubation times of 4 or 6 hours showed that GSPT1 was the only detectable protein degraded with greater than -1 log2 fold change (approximately 75% degradation). [2]
Genome-wide CRISPR-Cas9 screen in U937 cells identified genes and pathways that modulate response to CC-90009, including components of the CRL4CRBN complex, COP9 signalosome, neddylation pathway, as well as novel regulators such as ILF2/ILF3 (RNA splicing), TSC1/TSC2 (mTOR signaling), and GCN1/GCN2/ATF4 (integrated stress response). [1]
Knockout of ILF2 or ILF3 reduced full-length CRBN expression by altering CRBN mRNA alternative splicing, leading to diminished CC-90009-induced GSPT1 degradation and growth inhibition. [1]
Loss of TSC1 or TSC2 hyperactivates mTOR signaling and attenuates CC-90009 response by reducing GSPT1 recruitment to cereblon, decreasing GSPT1 degradation. Co-treatment with the mTORC1 inhibitor everolimus restored sensitivity. [1]
Knockout of GCN1, GCN2, ATF4, or DDIT4 protected cells from CC-90009-induced growth inhibition, confirming the role of the GCN2-mediated ISR pathway in mediating anti-AML activity. [1]

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Eragidomide-induced GSPT1 depletion causes acute myeloid leukemia (AML) apoptosis to occur quickly, which lowers leukemia stem cells (LSCs) and leukemia engraftment in large-scale primary patient xenografting of 35 different AML samples. Within AML blasts and LSCs, eragidomide activity is mediated by several tiers of signaling networks and pathways[1].

In HL-60 xenograft models of AML, treatment with CC-90009 significantly reduced tumor cell content in bone marrow. At 5 mg/kg BID for 5 days, there was a 54.0% reduction (p = 0.0013) of human CD33+/CD45+ tumor cells in bone marrow compared to vehicle. At 2.5 mg/kg BID for 10 days, there was a 71.5% reduction (p < 0.0001). [2]
In primary AML patient-derived xenograft models using NOD.SCID mice transplanted with 35 independent AML samples, CC-90009 treatment (2.5 mg/kg, IP, BID for 4 weeks) significantly reduced leukemic engraftment. Among 35 samples, 24 were responders (52-100% reduction in injected femur, 62-99% reduction in non-injected bones), 8 were partial responders (20-60% reduction), and 3 were non-responders (<20% reduction). [1]
GSPT1 degradation was observed in most AML xenografts within 24 hours after treatment with CC-90009. [1]
CC-90009 reduced the proportion of primitive CD34+ AML blasts enriched for leukemia stem cells in responding samples. In some samples, the absolute number of CD34+ cells in mouse bone marrow was significantly reduced. [1]
Serial limiting dilution assays in secondary recipients showed that CC-90009 decreased leukemia stem cell frequency in 3 out of 12 samples tested, with a ninefold decrease observed in a representative sample (AML3). Most samples (8 of 12) showed >50% reduction in absolute LSC numbers compared to controls. [1]
CC-90009 showed lesser inhibitory effect on normal cord blood CD34+ primitive cells in xenografts compared to AML CD34+ cells. [1]
All mice were healthy and tolerant to CC-90009 during the 4-week treatment period. [1]

Cell viability screening: AML cell lines, PBMCs from healthy donors, and THLE-2 cells were treated with CC-90009 at indicated concentrations for 3 days. Cell proliferation was assessed using the CellTiter-Glo assay. [1]
Primary AML patient sample assay: Bone marrow aspirates from AML patients were treated with DMSO or increasing concentrations of CC-90009 for 48-96 hours. Cells were stained with fluorescently labeled antibodies and Annexin-V, followed by flow cytometry to determine viable leukemic cell and lymphocyte counts. [1][2]
Western blot analysis: Cells (KG-1, U937, etc.) were treated with DMSO or CC-90009 at indicated concentrations and times. Where indicated, cells were pretreated with bortezomib or MLN4924 for 30 minutes. Whole cell extracts were prepared and subjected to immunoblotting with antibodies against GSPT1, CRBN, IKZF1, cleaved caspase-3, ATF4, phospho-eIF2α, and other proteins. [1][2]
Proteomics analysis: KG-1 cells were treated with DMSO or 100 nM CC-90009 for 4 hours and subjected to tandem mass tag proteomics analysis. For selectivity assessment, cells were treated with concentrations ranging from 0.20 μM to 3 μM for 4 or 6 hours. [1][2]
Cellular degradation assay: Cells were treated with CC-90009 for 4 or 20 hours, and GSPT1 levels were measured to determine EC50 and Dmax values. [2]
CRISPR-Cas9 screen: U937 cells stably expressing Cas9 were transduced with a lentiviral sgRNA library targeting ~19,000 protein-encoding genes. Cells were treated with 10 μM CC-90009 or DMSO for 9 days. Genomic DNA was isolated, sgRNA regions were amplified, and next-generation sequencing was performed to identify genes enriched or depleted by drug treatment. [1]
CRISPR competition assay: U937 or OCI-AML2 cells expressing Cas9 were infected with lentiviral vectors co-expressing RFP and sgRNAs targeting genes of interest, or GFP and control sgRNAs. RFP+ and GFP+ cells were mixed at 1:1 ratio and treated with DMSO or 10 μM CC-90009. The change in RFP+/GFP+ ratio was monitored by flow cytometry every 2 days. [1]
Immunoprecipitation assay: Cells expressing HA-tagged GSPT1 were treated with MLN4924 and DMSO or CC-90009. Anti-HA immunoprecipitates and whole cell lysates were analyzed by immunoblotting to assess GSPT1-CRBN interaction. [1]
RNA sequencing: U937-Cas9 cells with inducible expression of sgNT or sgILF3 were cultured for 7 days. RNA was extracted and subjected to mRNA sequencing to analyze differential gene expression and alternative splicing. [1]

HL-60 xenograft model: Female NOD.SCID mice were inoculated with HL-60 cells. Treatment was initiated 6 weeks post-inoculation. CC-90009 was administered by intraperitoneal injection at doses of 2.5 mg/kg or 5 mg/kg twice daily. One group received 5 mg/kg BID for 5 consecutive days (terminated on Day 7), another group received 2.5 mg/kg BID for 10 consecutive days (terminated on Day 11). The primary endpoint was percentage of human CD33+/CD45+ cells in bone marrow by FACS analysis. [2]
Primary AML patient-derived xenograft model: NOD.SCID mice were transplanted intrafemorally with primary AML cells. Starting 21 days post-transplantation, mice were treated with vehicle or CC-90009 at 2.5 mg/kg intraperitoneally twice daily for 4 weeks. GSPT1 degradation was assessed after 3 doses. Engraftment levels were measured in injected femur and non-injected bones by flow cytometry for human CD45+ and CD33+ cells. Serial limiting dilution assays were performed in secondary recipients to determine leukemia stem cell frequency. [1]
Formulation for in vivo studies: For mouse PK studies, CC-90009 was formulated in DMA/PEG400/5% dextrose in water (15:50:35) and administered by IV bolus at 2 mg/kg. For efficacy studies, the formulation was not specified in detail, but intraperitoneal administration was used. [2]

Mouse PK (CD-1 mice, IV 2 mg/kg): Clearance = 67.9 mL/min/kg, Volume of distribution = 1.00 L/kg, Mean residence time = 0.24 h. [2]
Rat PK (CD-IGS rats, IV 2 mg/kg): Clearance = 28.3 ± 0.7 mL/min/kg, Volume of distribution = 2.2 ± 0.5 L/kg, Mean residence time = 1.3 ± 0.3 h. [2]
Monkey PK (cynomolgus monkeys, IV 1 mg/kg): Clearance = 6 ± 1 mL/min/kg, Volume of distribution = 1.1 ± 0.1 L/kg, Mean residence time = 4 ± 1 h. [2]
Allometric scaling prediction for human: Clearance approximately 1.93 mL/min/kg, Volume of distribution approximately 1.48 L/kg. [2]
Metabolism: Metabolites of CC-90009 were investigated in hepatocytes from mouse, rat, monkey, and human, as well as in vivo in rat. No unique human metabolites were detected. [2]
Clinical PK (from phase 1 trial): Following intravenous administration, mean terminal half-life was approximately 9.3 hours. Upon 5 days of repeat dosing, 2- to 2.7-fold accumulation of total systemic exposure was observed across dosing schedules. Across all doses and schedules, geometric mean total clearance was 6.96 L/h, and volume of distribution was 61.69 L. Exposures increased with dose in each dosing schedule. [2]

hERG inhibition: CC-90009 showed modest activity against the hERG ion channel with IC50 = 5.3 μM. In a non-pivotal in vivo cardiovascular safety pharmacology study in cynomolgus monkeys, no drug-related cardiovascular effects were observed. [2]
CYP inhibition/induction: CC-90009 (up to 2.5 μM) did not cause inhibition or induction of CYP enzymes in vitro, except for weak inhibition of CYP2C9 (41% at 2.5 μM) and CYP2C19 (IC50 = 1.53 μM). [2]
Transporter inhibition: CC-90009 (up to 3 μM) did not inhibit P-gp, BCRP, MRP2, OAT1, OAT3, OATP1B1, OATP1B3, OCT2, or MATE2-K. [2]
Kinase panel: CC-90009 was screened against a panel of 255 kinases at 3 μM with no inhibition >20% observed. [2]
Receptor binding panel: At 10 μM, CC-90009 inhibited receptor binding >50% at only two receptors (M1 and M2) out of 80 human receptors, ion channels, and transporters tested. [2]
In vivo tolerability: In mouse xenograft studies, all mice were healthy and tolerant to CC-90009 during the 4-week treatment period at doses up to 5 mg/kg BID. [1][2]
Selectivity for AML cells: CC-90009 showed 2- to 5-fold greater growth inhibition of leukemic cells compared to normal lymphocytes from the same patients, and much less effect on PBMCs and THLE-2 cells compared to AML cell lines. [1][2]

[1]. Blood. 2021 Feb 4;137(5):661-677.

[2]. J Med Chem. 2021 Feb 25;64(4):1835-1843. .

[3]. https://www.cancer.gov/publications/dictionaries/cancer-drug/def/cereblon-modulator-cc-90009.

Erageldumine is a cereblon (CRBN) modulator, a component of the cullin 4-RING E3 ubiquitin ligase complex (CRL4-CRBN E3 ubiquitin ligase; CUL4-CRBN E3 ubiquitin ligase), which possesses potential immunomodulatory and pro-apoptotic activities. Upon administration, erageldumine specifically binds to CRBN, thereby affecting the activity of the ubiquitin E3 ligase complex. This leads to ubiquitination of certain substrate proteins and induces proteasome-mediated degradation of certain transcription factors, including the transcriptional repressors Ikaros (IKZF1) and Aiolos (IKZF3) in T cells. This reduces the levels of these transcription factors and modulates the activity of the immune system, potentially including T lymphocyte activation. Furthermore, it downregulates the expression of other proteins, including interferon regulatory factor 4 (IRF4) and c-myc, the latter playing a crucial role in the proliferation of certain cancer cells. CRBN is a substrate recognition component of the E3 ubiquitin ligase complex and plays a key role in the ubiquitination of certain proteins.

C22H18CLF2N3O4

461.8458

461.1

C, 57.21; H, 3.93; Cl, 7.68; F, 8.23; N, 9.10; O, 13.86

1860875-51-9

118647211

Off-white to light yellow solid powder

2.3

2

6

5

32

787

0

C1CC(=O)NC(=O)C1N2CC3=C(C2=O)C=CC(=C3)CNC(=O)C(C4=CC=C(C=C4)Cl)(F)F

PWBHUSLMHZLGRN-UHFFFAOYSA-N

InChI=1S/C22H18ClF2N3O4/c23-15-4-2-14(3-5-15)22(24,25)21(32)26-10-12-1-6-16-13(9-12)11-28(20(16)31)17-7-8-18(29)27-19(17)30/h1-6,9,17H,7-8,10-11H2,(H,26,32)(H,27,29,30)

2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide

CC-90009; CC 90009; Eragidomide; 1860875-51-9; CC-90,009; 2-(4-chlorophenyl)-N-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-2,2-difluoroacetamide; R76M2Z6366; CC90009

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 92~100 mg/mL ( 199.19~216.52 mM )

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)