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ERK signaling mediates resistance to immunomodulatory drugs in the bone marrow microenvironment

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Science Advances  04 Jun 2021:
Vol. 7, no. 23, eabg2697
DOI: 10.1126/sciadv.abg2697
  • Fig. 1 Genome-wide CRISPR-Cas9 screen identifies TRAF2 to be required for antimyeloma activity of Pom.

    (A) Volcano plot showing both positively and negatively selected genes in the CRISPR-Cas9 screen at day 21 after Pom treatment. Genes shown in red and blue represent positively and negatively selected genes, respectively. NS, not significant. (B) Normalized reads of sgTRAF2 from cells treated with either dimethyl sulfoxide (DMSO) control or Pom at the indicated time points. Veh, DMSO control. (C) Enrichment of TRAF2 and CRBN sgRNAs after Pom treatment. Each dot specifies one sgRNA. (D and E) Dose-dependent survival of Pom-treated (D) and Len-treated (E) MM.1S cells infected with individually cloned lentiCRISPR viruses targeting the selected gene candidates. Controls were null-targeting lentiCRISPR viruses. Error bars represent SEM (n = 3). IC50, half maximal inhibitory concentration. (F) Representative images of TRAF2 protein expression assessed by immunohistochemical staining of BM biopsies from six patients at time of diagnosis with disease sensitive to Len and at time of relapse with disease resistant to single-agent Len maintenance therapy. Scale bar, 20 μM. (G) Representative Western blot analysis of control and TRAF2 KO MM.1S cells treated with DMSO, 0.5 μM Pom, or 1 μM Len for 72 hours. Whole-cell lysates (WCLs) were collected and probed with indicated Abs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NT, non-targeting.

  • Fig. 2 TRAF2 KO inhibits apoptosis induced by IMiDs through enhancing noncanonical NF-κB and ERK pathway.

    (A and B) Representative Western blot analyses of control and TRAF2 KO MM.1S cells treated with DMSO or 1 μM Pom (A) or 1 μM Len (B) for 72 hours. WCLs were collected and probed with indicated Abs. (C) Percentage cell viability of control and TRAF2 KO MM.1S cells treated with 0.5 μM Pom for 72 hours. Cell viability was determined using CellTiter-Glo (CTG) cell viability assay. CRBN KO cells were analyzed as a positive control. Data are shown as means ± SEM. ***P < 0.001 by two-sided Student’s t test. (D) Gene set enrichment analysis plots of datasets identified comparing TRAF2 KO and wild-type signatures. NES, normalized enrichment score. FDR, false discovery rate. (E) Nuclear and cytoplasmic protein were extracted from MM.1S TRAF2 KO cells and immunoblotted with indicated Abs. Cyto, cytoplasmic; nuc, nuclear. (F) WCLs from control and TRAF2 KO MM.1S cells were probed for p-ERK, ERK, and TRAF2 by immunoblotting. The numbers under the bands of blots indicate band intensity normalized to control.

  • Fig. 3 Noncanonical NF-κB pathway mediates ERK pathway activation in TRAF2 KO cells.

    (A) Representative Western blot analysis of control and KO MM.1S cells. WCLs were collected and probed with indicated Abs. (B) Percentage cell viability of sgTRAF2 and/or sgp52 MM.1S cells treated with 0.5 μM Pom for 72 hours. Cell viability was determined using CTG assay. (C) KO efficiency of p100, p52, and TRAF2 in MM.1S cells was assessed by immunoblot analysis. (D) MM.1S cells were infected with lentivirus to constitutively express activated ERK2 and then treated with Pom (0 to 180 nM) for 72 hours. Cell viability was determined using CTG assay. Data in (B) and (D) are shown as means ± SEM. ***P < 0.001 by two-sided Student’s t test. OE, over-expressed; GFP, green fluorescent protein. (E and F) RNA-seq data from MM patient samples of 69 patients at first relapse on Len maintenance therapy. Enrichment scores for ERK pathway activation (E) and correlation between ERK and noncanonical NF-κB pathway activation (F) were analyzed.

  • Fig. 4 Combination of AZD6244 with IMiDs triggers synergistic antimyeloma activity.

    (A) Percentage cell growth of TRAF2 KO MM.1S cells after 3 days of treatment with Len (0 to 1 μM) and ERK inhibitor AZD6244 (0 to 1 μM). Cell growth was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. (B) Representative Western blot analysis of TRAF2 KO MM.1S cells treated for 48 hours with AZD6244 (0 to 3 μM), Len (0 to 3 μM), or both. WCLs were collected and probed with PARP Ab. (C to F) MM.1S cells expressing doxycycline-induced shTRAF2 were injected subcutaneously into CB-17 SCID mice (n = 5 for each group). When tumors reached 100 mm3, mice were randomized and treated with vehicle, Pom (2.5 mg/kg), AZD6244 (12.5 mg/kg), or both drugs for 3 weeks. (C) Macroscopic photographs after 21 days of therapy. (Photo credit: Jiye Liu, DFCI). Tumor volume (D) and body weight (E) were monitored over the indicated time period. Data in (D) and (E) are shown as means ± SEM. **P < 0.01 by two-sided Student’s t test. (F) Representative images of TRAF2 and p-ERK1/2 immunohistochemistry stain in tumor tissue sections from each group. Scale bars, 40 μM.

  • Fig. 5 BM microenvironment induces IMiD resistance through ERK pathway activation.

    (A and B) MM.1S cells were treated with indicated concentrations of Pom (A) or Len (B) in the presence or absence of BMSC. Cell growth was determined using MTT assay. (C) Percentage cell growth of MM.1S cells after 3 days culture with stromal cell supernatants (SC-sup). Cell growth (means ± SEM) was determined using MTT assay and normalized to medium control. SCs were from five patients with MM. (D) Percentage cell growth of MM.1S cells after 3 days of treatment with Pom (1 μM), SC-sup, or both. Cell growth was determined using MTT assay and normalized to the DMSO control group. (E) Immunoblot analysis of TRAF2 protein level in MM.1S cells cultured with SC-sup for 48 hours. SCs were from five patients with MM. (F) MM.1S cells were cultured for 0 to 24 hours in the presence or absence of SC-sup. WCLs were collected and probed with p-ERK1/2 and ERK1/2 Abs. The numbers under the bands of blots indicate band intensity normalized to control. Data in (A) to (D) are shown as means ± SEM. ****P < 0.0001 and **P < 0.01 by two-sided Student’s t test.

  • Fig. 6 Paracrine TNF-α in stromal cell supernatant induces IMiD resistance through proteasomal degradation of TRAF2.

    (A) Representative image of cytokine Ab array screening of SC-sup. Supernatant was collected after 24-hour culture with BMSCs and filtered by a 0.22 μM low-protein binding membrane. (B) Representative Western blot analysis of MM.1S cells treated with TNF-α or IL-6 for 24 hours. WCLs were collected and probed with TRAF2 Ab. (C) MM cell lines were treated with TNF-α (5 ng/ml) for 48 hours. Cell lysates were collected and blotted with TRAF2 Ab. (D) Immunoblot analysis of patient MM cells treated with TNF-α (5 ng/ml) for 48 hours. WCL was collected and probed with TRAF2 Ab. (E) Percentage cell growth of MM.1S cells after 5 days of treatment with Pom (0 to 3 μM) and/or TNF-α (0 to 3 ng/ml). Cell growth (means ± SEM) was determined using MTT assay. (F) MM.1S cells were treated with TNF-α (5 ng/ml) for 0 to 10 hours. WCLs were collected and probed with TRAF2 Ab. (G) MM.1S cells were treated with 0.5 nM BTZ or 2.5 nM CFZ for 2 hours, followed by treatment with TNF-α (10 ng/ml) for 24 hours. WCLs were collected and probed with TRAF2 Ab. (H) MM.1S cells were cultured with TNF-α (5 ng/ml) for 18 hours and then treated with MG132 (10 μM) for 6 hours. WCLs were collected and immunoprecipitated by anti-TRAF2 Ab and probed for polyubiquitinated protein and TRAF2. IP, immunoprecipitated. The numbers under the bands of blots indicate band intensity normalized to control.

  • Fig. 7 AZD6244 overcomes IMiD resistance induced by BM microenvironment.

    (A) Percentage cell growth of MM.1S cells after 5 days of treatment with Len (0 to 3 μM) and/or IL-6 (0 to 10 ng/ml). Cell growth (means ± SEM) was determined using MTT assay. (B) Representative Western blot analysis of MM.1S cells cultured for 72 hours in the presence of BMCS with AZD6244 (1 μM), Len (1 μM), or both. WCLs were collected and probed with anti-cleaved PARP, IKZF1, p-ERK, and ERK Abs. (C) Percentage MM.1S cell growth in cultures with or without SC-sup after 5 days of treatment with Pom (0 to 1 μM), AZD6244 (0 to 1 μM), or both. Cell growth (means ± SEM) was determined using MTT assay. (D) Percentage MM.1S cell growth in cultures with IL-6 (5 ng/ml) after 5 days of treatment with Pom (0 to 1 μM), AZD6244 (0 to 1 μM), or both. Cell growth (means ± SEM) was determined using CTG assay. (E) Percentage MM.1S cell growth in cultures with TNF-α (5 ng/ml) after 5 days of treatment with Pom (0 to 1 μM), AZD6244 (0 to 1 μM), or both. Cell growth (means ± SEM) was determined using CTG assay.

Supplementary Materials

  • Supplementary Materials

    ERK signaling mediates resistance to immunomodulatory drugs in the bone marrow microenvironment

    Jiye Liu, Teru Hideshima, Lijie Xing, Su Wang, Wenrong Zhou, Mehmet K. Samur, Tomasz Sewastianik, Daisuke Ogiya, Gang An, Shaobing Gao, Li Yang, Tong Ji, Giada Bianchi, Kenneth Wen, Yu-Tzu Tai, Nikhil Munshi, Paul Richardson, Ruben Carrasco, Yong Cang, Kenneth C. Anderson

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