Targeting an Inducible SALL4-Mediated Cancer Vulnerability with Sequential Therapy

Abstract

Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2'-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. SIGNIFICANCE: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.

Figure 1.. Engineered SALL4-partial dependency in SALL4-negative cancer cells.

(A) Western blot of SALL4 protein in GFP control (H1299-GFP) or exogenous SALL4-expressing H1299 (H1299-SALL4) cells 48 hours after transfection of shRNA negative control (NC) or SALL4 shRNA (SALL4), β-actin was used as normalized control. (B) Cell viability of GFP control or SALL4-expressing H1299 cells after the indicated treatments. (C) Flow cytometry plots showing Annexin/PI staining of GFP control or SALL4-expressing H1299 cells after the indicated treatments. (D) Percent apoptotic (Annexin⁺) and dead cells (Annexin⁻ /PI⁺) from (C). (E) Western blot of SALL4 protein in H661 cells 48 hours after transfection of negative control sh-NC or SALL4 shRNA. β-actin was used as normalized control. (F) Cell viability of H661 cells after indicated treatment. (G) Flow cytometry plots showing Annexin/PI following the indicated treatment. (H) Percent apoptotic and dead cells from (G). (I) Representative images of tumors harvested from mice 30 days following sub-cutaneous injection of H1299 cells expressing the indicated vectors. (J) Tumor weight and (K) body weight from indicated groups (N=5). n.s. means P>0.05, *P<0.05, **P<0.01, ***P<0.001, N=3.

Figure 2.. Upregulation of SALL4 by hypomethylating agents.

(A) SALL4 expression levels in cancer cell lines with either high- (>75%) and low- (<25%) CpG methylation at exon 1, data generated from CCLE database. (B) SALL4 expression levels in primary tumors from lung adenocarcinoma and squamous cell carcinoma, colon adenocarcinoma, hepatocellular carcinoma, and gastric adenocarcinoma with either high- (>75%) and low- (<25%) CpG methylation at exon 1, data generated from TCGA database. (C) Fold change of SALL4 expression measured by real-time PCR from patients’ bone marrow mononuclear cells after six cycles of 5-Aza treatment (C7D1/C1D1), GAPDH was used for a normalization control. (D) RNA-seq data of H1299 cells after treatment of 1 μM 5-Aza or DMSO, data generated from GSE5816. (E) mRNA and (F) protein levels of SALL4 in H1299 cells after 5 days treatment of DMSO or DAC. (G) Map of CpG islands tested within the first exon of SALL4 locus. (H) Methylation levels of CpG islands within the first exon of SALL4 genome in H1299 cells after indicated treatment. *P<0.05, ***P<0.001, N=3.

Figure 3.. ENT treatment decreased cell viability and induced cell apoptosis via inhibition of SALL4.

(A) Protein and (B) mRNA levels of SALL4 in H661 cells after the indicated treatment of ENT or DMSO control. (C) Pre-mRNA level of SALL4 in H661 cells after 48 hours treatment of ENT or DMSO control. (D) Cell viability of H661 cells after indicated treatment for 5 days. (E) Flow cytometry of H661 cells after indicated treatment. (F) Percent apoptotic and dead cells in (E). (G) Cell viability of ENT pre-treated H661 cells after transfection of negative control or SALL4 overexpression vector. (H) Flow cytometry of ENT pre-treated H661 cells after indicated treatment. (I) Percent apoptotic and dead cells in (H). *P<0.05, **P<0.01, ***P<0.001, N=3.

Figure 4.. MiR-205 was involved in ENT-induced SALL4 inhibition.

(A) miRNA sequencing results of H661 cells after 8 hours treatment with 2.5 μM ENT or DMSO. (B) miR-205 was up-regulated by ENT and predicted to target SALL4 by the Targetscan database. (C) Real-time PCR of miR-205 expression in H661 cells after 2.5 μM ENT or DMSO treatment for 8 hours. U6 was used as a normalization control. (D) ChIP-qPCR of the miR-205 promoter region and non-specific control region after 2.5 μM ENT treatment. IgG was used as a normalization control. (E) Sequence of wild-type SALL4 3’UTR with miR-205 binding site or mutant one without binding site. (F) Luciferase reporter result in 293T cells after transfection with the indicated vectors. (G) mRNA and (H) protein levels of SALL4 in H661 cells after transfection with miR-205 or non-specific control. (I) Cell viability of H661 cells after indicated treatment for 5 days. (J) Flow cytometry of H661 cells after the indicated treatment. (K) Percent apoptotic and dead cells in (J). (L) Cell viability of ENT pre-treated H661 cells after transfection of miR-205 or non-specific control. (M) Flow cytometry of ENT pre-treated H661 cells after indicated treatment. (N) Percent apoptotic and dead cells in (M). n.s. means P>0.05, *P<0.05, **P<0.01, ***P<0.001, N=3.

Figure 5.. DAC/ENT combination treatment targeted SALL4 negative cancer cells in culture.

(A) Schema of DAC and ENT combination treatment of H1299 or H661 cells. (B) Relative cell viability and IC₅₀ of DMSO or DAC pre-treated H661 cells following ENT therapy. (C) Relative cell viability and IC₅₀ of DMSO or DAC pre-treated H1299 cells following ENT therapy. (D) Schema of experiment design in (E&F). (E) Protein level of SALL4 in shNC or shSALL4 stable expressing H1299 cells after 5 days treatment of DMSO or DAC. (F) Percentage of IC₅₀ against ENT for indicated H1299 cells, IC₅₀ of DMSO treated group was used as normalization control. (G) Schema of experiment design in (H&I). (H) Protein level of SALL4 in H1299 cells after 7, 14, or 21 days of DMSO or DAC treatment. (I) Percentage of IC₅₀ against ENT for indicated time point of H1299 cells, IC₅₀ of DMSO treated group was used as normalization control. n.s. means P>0.05, *P<0.05, **P<0.01, ***P<0.001, N=3.

Figure 6.. DAC/ENT combination treatment targeted SALL4 negative cancer cells in vivo.

(A) Schema of sequential drug treatment in a murine xenograft model. (B) Tumor growth measured at different time points. (C) Tumor volume measured at the end point from the indicated treatment groups. (D) Image of tumors harvested from mice with H1299 cells xenotransplantation after indicated treatment. (E) Tumor weights and (F) body weights from indicated groups. n.s. means P>0.05, *P<0.05, **P<0.01, ***P<0.001.