Cancer Genomics

Multiple myeloma is defined by aberrant monoclonal proliferation of plasma cells in the bone marrow. This remains an incurable disease despite advances in treatment. Key genetic and epigenetic alterations that drive MM pathogenesis are known, but we are endeavoring to create a comprehensive profile of affected cellular pathways. We integrate whole-genome and whole-exome sequencing data with single-cell RNA sequencing (scRNA-seq) data across multiple treatment stages to 1) assess differential pathway enrichment between tumor subpopulations, 2) trace the clonal evolution of dominant disease mechanisms, and 3) investigate signaling interactions between surrounding cell types. Our findings advance the understanding of how MM tumor subpopulations differentially regulate cellular pathways and interact within the tumor microenvironment.(1)

We also use genomics to attempt to overcome a major limitation in the development of immunotherapies: discovery of optimal candidate targets.  These require both high expression in tumor cells as well as stringent tissue specificity. To identify potential myeloma and AML-specific target antigens, we perform unbiased searches for genes with specific expression in plasma and/or B cells using single cell RNA-seq (scRNA-seq), single cell Assay for Transposase-Accessible Chromatin (scATAC-seq), single cell proteomics, whole exome sequencing and bulk RNA-sequencing. Our strategies have enabled the discovery of myeloma-associated therapeutic target candidates, including tumor-specific neoantigens and tumor-associated antigens. Similar studies using AML samples are ongoing. These studies are enabling us to develop novel treatment options (i.e., CAR-T, bispecific antibody, vaccine, etc.) for patients with myeloma or AML.

1. Text in this paragraph first appeared in its entirety in: Wang, et al., Blood. 134 (Supplement 1): 364

Immune Evasion

We are also studying the molecular mechanisms that underlie relapse after bone marrow or stem cell allogeneic transplantation which is currently the only curative therapy for AML. Using mouse models and human specimens we strive to identify and characterize the genomic and transcriptional variants that provide long-term, relapse-free survival. Using the former we generated allogeneic T cell-resistant (alloTR) murine AML cells in vivo by applying immunological pressure via serial alloHSCTs with MHC-matched, mHA-mismatched alloHSCT. We are searching within the transcriptome of these alloTR cells for dysregulation of minor histocompatibility antigens, immune-related genes or other metabolic pathways.

In patients, we recently found several genes involved in major histocompatibility class II (MHCII) antigen processing and presentation are significantly downregulated after alloHSCT but not after chemotherapy. Significantly, we find that treating cell lines or patient cells with IFN-γ rapidly re-induces MHCII expression and re-sensitizes the cells to cytolysis in mixed lymphocyte reactions.

Bispecific antibodies bypass MHC restriction by creating an artificial T cell synapse between effector T cells and target cells. We study samples from patients with AML who were treated with bispecific antibodies targeting CD123 and CD3 (flotetuzumab, NCT02152956) or CD33 and CD3 (AMV564, NCT03144245).  Using 21-color flow cytometry we simultaneously characterize tumor markers and the T cell subsets in the samples.  Likewise, using a Luminex assay, we assess the concentration of 18 different serum cytokines, some of which (IL-6 and interferon-γ) play a role in cytokine release syndrome.  This led us to demonstrate that MHCII can be upregulated on human MHCII-negative blasts both in vitro and in mice by either flozetuzumab or chimeric antigen receptor T (CART) cells in an IFNγ-dependent manner.  Since MHCII upregulated blasts were killed more effectively by third-party mismatched human CD4 T cells than were untreated controls, this insight may provide a potential means to overcome relapse. We are examining this in a phase 2 clinical trial of flotetuzumab in patients with relapsed or refractory AML after allogeneic stem cell transplant (NCT04582864).

We also collaborate with other academic or industry partners to assess proprietary molecules in preclinical studies that have the potential to upregulate MHCII.