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Research Highlights

AACR-SU2C Research Highlights

High tumor mutation burden is associated with T cell dysfunction

Title: The T cell differentiation landscape is shared by tumour mutations in lung cancer
Grant: SU2C-American Lung Association-LUNGevity Lung Cancer Interception Dream Team
SU2C Grantee: Charles Swanton, MBBS
Citation: Nature Cancer 1: 546-561

Major impact: Lung cancer tissues with a higher tumor mutation burden (TMB) have a preponderance of dysfunctional T cells. This skewing towards T cell dysfunctionality may be associated with poorer survival outcomes. Characterization of these dysfunctional and terminally differentiated T cells may be used to develop new therapeutic strategies to mobilize the immune system against cancer.

Dr. James Reading, who led the study between the laboratories of Prof. Sergio Quezada and Prof. Charles Swanton explains:

“We charted the rise and fall of the immune response to lung cancer. Our bodies naturally recognise tumours as being foreign due to mutations such as those caused by smoking. When the immune system targets these mutations, it wages a war against cancer, but over time our killer cells become exhausted and their resources drained. By understanding how this process of mutation-driven immune exhaustion takes place, our work could lead to new ways of resurrecting the immune system, in particular by sustaining a fresh army of killer cells to strike at these mutations, which we believe are the Achilles heel of cancer.”

Dr. James Reading

Summary:  A high TMB has been associated with an increased likelihood of response to immune checkpoint inhibitors. On the other hand, antigens’ continuous presence may lead to constant or repetitive[MN1] T cell stimulation and eventual dysregulated T cell function. To explore this possibility in early stage lung cancer, the authors analyzed tumor samples from untreated patients with Stage I-IIIA disease (as part of the TRACERx study). They found that tumor tissue, especially regions with higher TMB, contained a higher percentage of dysfunctional  (Tdys)  and terminally differentiated T cells (TDT) than non-cancerous tissue. Consistent with dysfunction, Tdys cells expressed the immune checkpoint proteins ICOS and CTLA-4, while TDT cells were characterized by expression of Eomes and low levels of IL-7 receptor. Single-cell RNA sequencing data analyses showed that the Tdys and TDT subsets had significant transcriptional similarity with dysfunctional T cells that are observed after persistent antigen exposure. Flow cytometry and effector gene analyses suggested that these cells remained functional by virtue of granzyme B, IFNG, CD40LG expression (in CD4 Tdys cells) and TNFRSF9 expression (in CD8 Tdys cells). The abundance of dysfunctional T cells appeared to be associated with a genetic signature termed TL-DS that resulted from the loss of two proteins, TCF7 and LEF, and from differentiation skewing. Analyses of an independent set of 900 lung cancer samples indicated that the TL-DS genetic pattern was associated with higher TMB and poorer outcomes. In addition, the TL-DS gene signature was associated with survival in nine other cancer types. The findings in this study suggest that when early differentiated T cells encounter antigens in early stage cancers, a higher number of mutations may cause them to morph into dysfunctional states. Since these antigen-exposed T cells express high levels of checkpoint proteins, they may be susceptible to checkpoint inhibitors.

Precursor Multiple Myeloma already show signs of a compromised immune microenvironment

Title: Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma
Grant: SU2C Multiple Myeloma Dream Team Grant
SU2C Grantee: Irene M. Ghobrial, MD; Gad Getz, PhD; Rafael Fonseca, MD; Mark Bustoros, MD
Citation: Nature Cancer 1: 493–506

scRNA-seq on precursor myeloma

Major Findings/Summary: Currently, individuals with precursor stages of multiple myeloma – monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM) –  are not treated until after they have progressed to multiple myeloma (MM).  Knowing the characteristics that portend a higher likelihood of progression to MM may help in monitoring individuals with MGUS and SMM and in developing strategies to prevent worsening of their conditions.

To help address this need, the authors analyzed blood and bone marrow (BM) samples from patients with MGUS, low or high-risk SMM or MM, and healthy individuals.  Profiling of scRNA seq and CyTOF data showed that changes in the BM immune cell composition (enrichment in NK and T cells and general decrease in pre-B cells and CD14+ monocytes) occured as early as the MGUS stage. In addition, a depletion of memory CD8+ T cells was observed in the SMM and MM stages. Results from in vivo studies with transplantable myeloma mice with different memory cell differentiation stages, strongly suggest an important immunosurveillance role of these memory cells against MM. CD14+ monocytes from MGUS, SMM, and MM patients had significantly lower surface expression of the MHC Class II surface receptor HLA-DR than CD14+ monocytes from healthy donors. The authors confirmed that this downregulation was induced in the presence of multiple myeloma cells. This decrease in HLA-DR may be due, in part, to increased expression of the E3 ubiquitin protein ligase MARCHF1/MARCH1, and decreased expression of the ER-resident protein VAMP associated protein A (VAPA) by CD14+ monocytes. Treating CD14+ monocyte/MM cocultures with anti-MARCHF1/MARCH1siRNAs rescued HLA-DR surface expression. CD14+HLA-DRlow cells play a role in MM cell proliferation and suppression of T-cell activation.


Precancerous lung lesions evade the immune system

Title: Immune surveillance in clinical regression of pre-invasive squamous cell lung cancer
Grant: SU2C-American Lung Association-LUNGevity Lung Cancer Interception Dream Team
SU2C Grantee: Sam M. Janes, MBBS, PhD, University College London
Citation: Cancer Discovery; published OnlineFirst July 20, 2020

Major Finding: The Team demonstrated the important role of the immune system in helping determine whether or not precancerous lung lesions will progress to cancer. Their findings may pave the way for new treatment strategies to treat or prevent early-stage lung cancer.

Figure 1b, 1c

Summary: Only half of high-grade lung carcinoma in situ (CIS) lesions will progress to cancer within two years, whereas a third will spontaneously regress. The authors hypothesized that immune system surveillance can prevent squamous carcinoma lesions from developing into cancer. A deep-learning approach and immunohistochemical analyses of samples from patients with pre-invasive lesions showed that progressive lesions had more infiltrating lymphocytes, particularly CD8+ cytotoxic T-cells, than regressive lesions. Nevertheless, “immune hot” progressive lesions were also observed, portending mechanisms of immune evasion. Mutations and copy number aberrations in genes critical for antigen presentation and processing and for immunomodulation of T cell responses were more prevalent in progressive than regressive samples.  In addition, differential expression analyses showed that TNFSF9 (4-1BBL) was significantly downregulated in progressive lesions. TNFSF9 was shown to promote the activation of T cells and NK cells.

CRISPR screening uncovers potential combinations with BCMA-targeted immunotherapies in multiple myeloma

Title: CRISPR-based screens uncover determinants of immunotherapy response in multiple myeloma
Grant: SU2C Innovative Research Grant
SU2C Grantee: Martin Kampmann, PhD
Citation: Blood Adv 4: 2899–2911

Major Finding: CRISPR interference- and CRISPR activation-based screening approaches were used to identify pathways that control cell surface expression of the multiple myeloma immunotherapy antigen B-cell maturation antigen (BCMA). Targeting these pathways suggest promising combinatorial strategies with BCMA-targeted immunotherapies.

Martin Kampmann, PhD
Martin Kampmann, PhD

Summary: BCMA-targeted immunotherapy agents have shown some promise in the clinic, although resistance and relapse have also been observed. One of the mechanisms of resistance is antigen escape as a result of BCMA downregulation or loss. The authors found that knocking down the genes from the gamma-secretase, HDAC7, or Sec61 pathways or blocking the activity of these proteins using small molecules was effective in inducing an increase in BCMA levels at the cell surface. Combining a gamma-secretase or Sec61 inhibitor with a BCMA-targeted antibody-drug conjugate (ADC) increased the potency of BCMA-targeted ADC. Taking it one step further, the authors used CRISPR technology to identify genes or cellular pathways that influenced the susceptibility of MM cells to BCMA-targeted CAR-T cells. Knocking down ICAM1, a protein involved in T cell activation, and DFFA, a protein involved in cell death, reduced sensitivity to these CAR-T cells. On the other hand, knocking down genes that encode for proteins called diacyglycerol kinases increased sensitivity to the CAR-T cells. Thus, the authors demonstrated that CRISPR-interference and CRISPR-activation are complementary CRISPR screening approaches that can be used to identify potential combinatorial treatments.

Off-the-shelf therapeutic cells can escape host immune rejection

Title: Engineered off-the-shelf therapeutic T-cells resist host immune rejection
Grant: SU2C Meg Vosburg T-Cell Lymphoma Dream Team Grant
SU2C Grantees: Helen Heslop, MD; Malcolm Brenner, MD, PhD; Maksim Mamonkin, PhD
Citation: Nature Biotechnology; published OnlineFirst July 13, 2020 

Major Finding: Engineering off-the-shelf T-cells to express an alloimmune defense receptor (ADR) protects them from rejection by the immune system without compromising their cancer-killing activity.

The Impact: One of the most promising immunotherapeutic strategies is the use of therapeutic T-cells. Unfortunately, it is more expensive than other therapies, partly because of the more complicated manufacturing process which includes isolating T cells from patients, genetically engineering them, and propagating them to numbers sufficient for treatment.

Summary: Off-the-shelf T cells can be as effective as therapeutic T cells (such as CAR-T cells) minus the cost.  However, because off-the-shelf T cells are generated from healthy donors, the patient’s immune system “sees” these T cells as foreign and sets out to destroy them. To get around this, the authors engineered off-the-shelf T cells to express a protein called 4-1BB-specific alloimmune defense receptor (ADR).  These ADR-expressing T cells were able to avoid attack from activated, but not resting, NK and T cells. Importantly, the authors demonstrated that engineering CD19 CAR-T cells and GD2 CAR-T cells with the ADR does not blunt the CAR-T-cells’ tumor cell killing activity in cell culture and in animal experiments. The ADR-expressing T cells were able to counteract the host immune system and thus persist longer in animal models of leukemia and neuroblastoma, resulting in better outcome.

New genetic model increases ability to predict risk of progression from SMM to MM

Title: Genomic profiling of smoldering multiple myeloma identifies patients at a high risk of disease progression
Grant: SU2C Multiple Myeloma Dream Team Grant
SU2C Grantees: Irene M. Ghobrial, MD; Gad Getz, PhD; Chip Stewart, PhD
Citation: Journal of Clinical Oncology 38: 2380-2389

Major Finding: There is an urgent need to accurately identify patients with precursor stage myeloma, smoldering multiple myeloma (SMM), who are at risk for disease progression and who could benefit from early treatment. The authors developed a genetic model that can be combined with a clinical model to better predict the likelihood that a patient with SMM will progress to multiple myeloma (MM).

Irene M. Ghobrial, MD

Summary: The authors analyzed samples from 85 untreated SMM patients and found that patients who had MYC gene aberrations had the shortest time to progression. Patients who had MAPK and DNA repair pathway alterations also had a relatively shorter time to progression than patients who did not have such alterations. Genetic aberrations in the MYC, DNA repair, and MAPK pathways were found to be associated with higher bone marrow infiltration at the time of diagnosis. The authors developed a genetic model that integrates information on these pathway aberrations to predict the likelihood of disease progression and tested this genetic model on an independent set of 72 SMM patient samples. Patients with any of these three aberrations had a higher risk of progression. Combining this new genetic model with the traditional clinical model better predicted a patient’s risk of progression than using the clinical model alone. Additional studies with larger patient cohorts are recommended to assess whether studying other genetic features such as deletion in the 1p or 8p chromosomal arm can be used to help predict the likelihood of progression from SMM to MM. Given that most of the critical mutations in MM can be observed at the SMM stage, genomic profiling of patient’s tumors can potentially be performed at the time of SMM diagnosis. This strategy may improve the ability to identify patients who are likely to progress to MM and may benefit from early treatment.

Spatial profiling of immune cells to understand lung cancer development and relapse

Title: Geospatial immune variability illuminates differential evolution of lung adenocarcinoma
Grant: SU2C-American Lung Association-LUNGevity Lung Cancer Interception Dream Team Grant
SU2C Grantee: Charles Swanton, MBBS
Citation: Nature Medicine 26: 1054-1062

Major Finding: Deep-learning integration of digital pathology data can be used to understand how lung cancer cells escape the defensive action of the immune system.  Analyses of data from more than 1000 lung cancer patient samples showed that patients whose tumors had more than one immune cold region had a higher risk of relapse. This work reinforces the interplay between cancer cells and the immune system in influencing the progression of cancer.

Charles Swanton, MBBS
Charles Swanton, MBBS

Summary: Cancerous cells persist, at least in part, because they are able to escape the immune system’s attack. To interrogate the role of the immune system in lung cancer development, the authors used deep-learning methods to analyze histology data from 100 patients with lung cancer from the TRACERx cohort, and spatially mapped cancer cells, immune cells, and stromal cells. To further validate their findings, samples from an independent set of 970 lung cancer patients were analyzed. The data suggest that patients who had lung cancer tissue that had multiple immune cold regions had a higher risk of relapse, regardless of the size and stage of their lung cancer. To help elucidate the mechanism of immune cell exclusion in certain tumor regions, the authors studied the location of the stromal cells in reference to cancer cells. Using fractal dimension as a spatial measure to quantify the complexity of the interaction between cancer cells and stromal cells, the authors found that fractal dimension was higher in immune cold regions than in immune hot regions. This suggests that the degree of interaction between cancer cells and stromal cells affects the ability of immune cells to infiltrate cancer tissue. This study demonstrates how deep learning algorithms can be used to leverage pathology data, characterize the spatial relationships between cancer cells, stromal cells, and immune cells, and make predictions regarding lung cancer progression and relapse.

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