# A heterodimer of hemoglobin identifies theranostic targets on brain‐metastasizing melanoma cells

## TL;DR

A hemoglobin heterodimer in the brain microenvironment kills melanoma cells by targeting BRD4 and IRS2 proteins, and combining inhibitors of these proteins shows promise in treating brain metastases.

## Contribution

Combining BRD4 and IRS2 inhibitors mimics the anticancer effects of a hemoglobin heterodimer in brain-metastasizing melanoma cells.

## Key findings

- A combination of BRD4 and IRS2 inhibitors synergistically kills brain-metastasizing melanoma cells.
- The BRD4/IRS2 inhibitor cocktail reduces malignancy in multiple melanoma cell lines.
- RNAseq analysis shows TNF and apoptotic signaling pathways are enriched after treatment.

## Abstract

Cancer microenvironments encompass both cancer‐promoting and cancer‐restraining factors. If these factors cancel each other, cancer dormancy may ensue. In search of microenvironmental factors that keep dormant lung‐metastasizing neuroblastoma cells and brain‐metastasizing melanoma cells (BMMC) in check, we identified the beta subunit of hemoglobin and a heterodimer of alpha and beta chains of hemoglobin (α/β dimer) in the lung and brain microenvironments, respectively, as anti‐metastatic factors. A previous study demonstrated that the α/β dimer triggers programmed cell death of BMMC and downregulates the expression of BRD4, GAB2, and IRS2 proteins, which perform essential functions in tumorigenesis and progression. The working hypothesis of the present study is that in addition to its tumoricidal function, the α/β dimer serves as a pathfinder for the identification of therapy targets for BMMC. We, therefore, employed small‐molecule inhibitors of Bromodomain‐containing protein 4 (BRD4), GRB2‐associated‐binding protein 2 (GAB2), and Insulin receptor substrate 2 (IRS2) as potential anti‐BMMC agents. A combination of sub‐lethal concentrations of BRD4 and IRS2 inhibitors synergistically arrested BMMC at the subG1 phase of the cell cycle and killed more than 70% of BMMCs. The BRD4/IRS2 inhibitor cocktail (designated hereafter as BRIRi) moderated the malignancy of BMMC lines from four different human melanomas. Preliminary results suggest that the BRIRi modulated “cold” BMMC to “hot” ones. Among the top enriched functions of differentially expressed genes identified by RNAseq of BRIRi‐treated versus control BMMC, TNF and apoptotic signaling pathways were observed. We propose that co‐targeting BRD4 and IRS2 offers a promising approach for treating BMMC.

What's new?

Hemoglobin‐derived molecules have been shown to exhibit innate anti‐cancer functions similar to antimicrobial peptides and proteins. Previously, the authors identified a heterodimer of the alpha and beta chains of hemoglobin in the brain microenvironment that killed brain‐metastasizing melanoma cells by targeting BRD4 and IRS2 proteins. Here, they found that small‐molecule inhibitors of BRD4 and IRS2, in combination, mimicked the effects of the hemoglobin alpha/beta heterodimer and restricted the malignant phenotype of brain‐metastasizing melanoma cells. Taken together, the findings highlight the potential of innate anticancer factors to help identify therapy targets on cancer cells.