Rrunt-related transcription factor (RUNX)

AI-10-104 (0-10 μM) inhibits RUNX by dissociating the RUNXs-IKZFs complex, enhancing the antiproliferative effect of lenalidomide (CC-5013) and its cytotoxicity against myeloma cells [1].

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RUNX inhibition dissociates the IKZFs–RUNXs complex and potentiates the cytotoxic effect of lenalidomide in myeloma [1]
To assess whether chemical inhibition of RUNXs results in changes in RUNXs–IKZFs interaction, we tested the pan-RUNX inhibitor, AI-10-104. AI-10-104 interferes with the association of RUNX with CBFβ, thereby leaving RUNX in an auto-inhibited state. AI-10-104 induced a dose-dependent dissociation of CBFβ and IKZF3 from RUNX1 (Fig. 4a) and RUNX3 (Fig. 4b). Importantly, treatment of cells with increasing concentrations of AI-10-104 resulted in increasing RUNX1 and RUNX3 dissociation from IKZF3 (Fig. 4a, b). Similar data were produced when IKZF1 was pulled down (Fig. 4c, d). AI-10-104 did not displace RUNX1–IKZF1/3 interaction at the AD–ID interface (Supplementary Fig. 3a), hence it is possible that AI-10-104 dissociates the RUNX1–CBFβ complex, resulting in a conformational change ultimately affecting IKZFs interaction.

Next, we tested whether the inhibition of RUNXs could enhance the cytotoxic effect of lenalidomide. To this end, we treated five MM cell lines with combination doses of lenalidomide and AI-10-104. Low doses of lenalidomide had a modest effect on MM cell proliferation (Fig. 4e), however, combination of the two drugs induced an overall proliferation defect in the MM cell lines tested (Fig. 4e). Combination index (CI) calculation revealed that the MM cells expressing low RUNX1 (NCI-H929, MM1S, and U266, Supplementary Fig. 2b) were particularly sensitive to the drug combination, revealing a synergic effect at sub-micromolar dosage (Fig. 4f). CI calculation for the lenalidomide-insensitive cells lines, OPM-1 and RPMI-8226 cells, also displayed synergy, although a higher concentration of both drugs was required. In line with this, combination of AI-10-104 and lenalidomide downregulated the myeloma oncogenes MYC and IRF4 (Supplementary Fig. 3b).

Notably, when used as single agent, AI-10-104 displayed a moderate effect on cell proliferation (Fig. 4e), in line with the genetic evidence that ablation of RUNX1 and RUNX3 had no effect on proliferation of MM cells (Fig. 3h and Supplementary Fig. 2c).

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RUNX inhibition potentiates the transcriptional response induced by lenalidomide [1]
To understand the molecular basis for the efficacy of the combination regiment of lenalidomide and RUNX inhibition in treating myeloma, we profiled gene expression changes by RNA sequencing of OPM-1 cells (Supplementary Tables 2, 3 and 4). To this end, we treated cells with 0.1 μM lenalidomide or 1 μM AI-10-104 and both in combination. As shown previously, single treatments at the indicated concentrations are not effective in blocking OPM-1 proliferation (Fig. 4e). Notably, low doses of lenalidomide resulted in deregulation of 69 genes as compared to DMSO counterparts while RUNX inhibition resulted in changes in 38 genes. Importantly, the combination of drugs promoted deregulation of 105 genes (Fig. 5a, b and Supplementary Fig. 5) with upregulation of 60 genes specifically (Fig. 5b). Gene ontology (GO) analysis revealed that the most consistent signatures upregulated by combining lenalidomide and RUNX inhibitor were those associated with interferon signaling, immune response, and inflammatory response (Fig. 5c). Similarly, gene set enrichment analysis (GSEA) revealed significant expression of interferon response gene signatures (Fig. 5d). Specifically, OPM-1 cells treated with the combination of drugs upregulated genes related to interferon (IFN) response relative to either agent alone, including HLA-DQA1, HLA-DRB1, CD74, OAS2, ISG15, NMI, IFIH1, and EPSTI1 (Fig. 5e).

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Chemical inhibition of RUNXs potentiates lenalidomide toxicity in primary MM cells [1]
We evaluated primary myeloma samples for their sensitivity to combinatorial therapy consisting of RUNX inhibitor and lenalidomide. Primary myeloma cells were isolated from the iliac crest of patients and subjected to CD138-positive purification. Treatment of diagnostic and relapsed myeloma samples (Fig. 6b) with AI-10-104 or lenalidomide alone resulted in minimal or no change in cell viability when compared to DMSO (Fig. 6a). The combinatorial treatment of AI-10-104 and lenalidomide instead displayed a significant inhibitory effect on the viability of primary myeloma samples. These data are in line with our previous observations in MM cell lines, where single treatment at low doses of lenalidomide or AI-10-104 did not reduce cell viability as compared with DMSO (Fig. 4e). Again, combination of the two drugs effectively reduced cell viability. Of note, the cytotoxic effect of the lenalidomide and AI-10-104 combination regiment was not dependent on patient treatment history (Fig. 6b).

Importantly, treatment of normal human hematopoietic cells with AI-10-104 resulted in an average IC50 of ~15 μM [36, 37], which greatly exceeded the toxic concentration for MM cell lines and MM primary cells. Thus, RUNX inhibition in myeloma patients should not result in toxic adverse effects on normal hematopoietic stem and progenitor cells. Our data suggest that a therapeutic window may exist for the combinatorial therapy of AI-10-104 derivatives and lenalidomide in MM patients.

Western Blot Analysis[1]
Cell Types: HEK293T
Tested Concentrations: 0-10 μM
Incubation Duration:
Experimental Results: Decreased levels of IKZF3 and CBFβ.

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Cell Proliferation Assay[1]
Cell Types: OPM-1 cells, KMS-11 and U266 cells
Tested Concentrations: 1 μM
Incubation Duration:
Experimental Results: Maintained the cell viability, inhibited proliferation of OPM-1 cells in a moderate effect.

[1]. RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation. Leukemia. 2019 Aug;33(8):2006-2021.

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. [1]

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Introduction of proteasome inhibitors (bortezomib and carfilzomib) and IMiDs have changed the treatment paradigm for myeloma. However, myeloma remains incurable and new treatments are currently being studied. Here, we show that inhibition of RUNXs via the small molecule inhibitor AI-10-104 potentiates the cytotoxic effect of lenalidomide in MM cells. RUNX1 downregulation was previously suggested as a possible therapeutic avenue for the treatment of MM. Furthermore, previous evidence points out a role for RUNX2 in promoting expression of multiple metastatic genes and favoring homing of MM cells to the bone. Our data suggest that RUNX proteins are dispensable with regard to the proliferation potential of MM cells, and the drugs that achieve pan-inhibition of the RUNX proteins might act as an intervention point for the treatment of MM, particularly in combination with low doses of lenalidomide (Supplementary Fig. 6).

C14H10F3N3O2

309.243313312531

1881276-00-1

Typically exists as solids at room temperature

O(C(F)(F)F)C1C=CC2=C(C=1)N=C(C1N=CC=CC=1OC)N2

AI-10-104; 1881276-00-1; SCHEMBL17525962; OIIHJPSDBLPWEL-UHFFFAOYSA-N;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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