Identification of Thrombospondin-1 As a Novel Substrate of Cereblon Responsible for IMiDs-Induced Venous Thromboembolism

Immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide, are key drugs for the treatment of multiple myeloma (MM) and myelodysplastic syndrome(MDS) with chromosome 5q deletion[del(5q)].IMiDs modulate the ubiquitination of intracellular proteins by E3 ubiquitin ligase and alter the stability of target proteins via proteasomal degradation. IMiDs directly bind to cereblon (CRBN), a substrate receptor for the E3 ubiquitin ligase complex, and retarget the enzyme complex toward neo-substrates such as IKAROS family zinc finger 1/3 (IKZF1/3) and casein kinase 1α (CK1α) in MM and MDS with del(5q), respectively, which underlies their clinical efficacy in these disorders. Thus, the identification of cell specific neo-substrates should be a critical step to understand IMiDs-mediated pleiotropic effects.

Clinically, IMiDs cause venous thromboembolism (VTE); however, the molecular machineries how IMiDs increase the risk of VTE still remains elusive. We, therefore, hypothesized that there is an unknown substrate for CRBN that induces thrombosis in megakaryocytes and platelets. To identify possible molecules that could interact with CRBN in human megakaryocytes, we performed screening analysis. Human CD34⁺hematopoietic stem/progenitor cells (HSPCs) from cord blood were cultured in a serum-free megakaryocytic condition for 7 days as previously reported. Most of the CD34⁺HSPCs were differentiated into CD41⁺CD42b⁺mature megakaryocytes, and we extracted human megakaryocytic protein. Cellular lysates from CD34⁺HSPCs-derived megakaryocytes and FLAG-tagged CRBN-overexpressing HEK293T cells were mixed with or without lenalidomide. CRBN-binding proteins were subsequently pulled down using anti-FLAG antibody, and subjected to mass spectrometry analysis. Mass spectrometry analysis revealed that THBS1 was coimmunoprecipitated with CRBN, and its interaction was decreased in the presence of lenalidomide. We therefore hypothesized that THBS1 could be a neo-substrate for CRBN. Consistent with the screening results, we found that IMiDs increased THBS1 protein in Meg-01 cells, a human megakaryoblastic leukemia cell line in a dose and time dependent manner, without affecting mRNA level. Proteasome inhibitor increased THBS1 level in Meg-01 cells, suggesting that THBS1 should be physiologically degraded via proteasome. The immunoprecipitation experiments showed CRBN and THBS1 conjugation, which was inhibited in the presence of Lenalidomide in vitro. Furthermore, we confirmed an increase in THBS1 protein level in the bone marrow and plasma of MM patients treated with IMiDs-containing regimen, suggesting that IMiDs actually induce the accumulation of THBS1 in vivo. These results collectively suggest that THBS1 is a neo-substrate for CRBN and IMiDs induces the accumulation of THBS1 through the inhibition of proteasomal THBS1 degradation in human.

THBS1 is reported to competitively inhibit ADAMTS13 activity of von Willebrand Factor (vWF) multimer cleavage, leading to increased level of large vWF multimers. Since large vWF multimers promote thrombus formation and several studies showed increased plasma level of THBS1 correlates with the incidence of VTE, we tested whether the accumulation of THBS1 induced by IMiDs treatment could affect the size of vWF multimer in MM patients. vWF multimer analysis revealed the large vWF multimers increased in plasma of MM patients undergoing IMiDs treatment. These results collectively suggest that IMiDs-induced accumulation of THBS1 should be one of the molecular machineries for IMiDs-induced VTE in human.