CRISPR versus acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is an aggressive form of cancer arising from malignant transformation of immature cells that were otherwise fated to become white blood cells, or lymphocytes. ALL occasionally affects adults but is more commonly a pediatric cancer: children under the age of 5 have the highest risk of being affected. Upon diagnosis, it is possible to treat ALL with an aggressive regimen of multiple chemotherapy drugs that is successful for over 80% of patients. Unfortunately, when tumors reappear after initial treatment, in relapse, the cases become extremely difficult to treat. Additionally, the cellular landscape of ALL in relapse has a high degree of genetic heterogeneity and variability between different patients. Oncologists and cancer biologists suspect it is this genetic complexity that makes ALL in relapse especially hard to fight in the clinic. 

In a study published in Nature Cancer last year, Dr. Jessie Brown and colleagues set out to improve outcomes for patients with ALL by clarifying how mutational complexity in relapsed tumors interacts with chemotherapy drugs to resist treatment. To this end, the team first employed extensive genetic sequencing on ALL samples collected upon diagnosis, remission, and relapse in order to identify the mutational landscape that distinguishes relapse tumors from the others. Samples were collected from 175 patients in total – 149 from pediatric cases and 26 from adults. Next, in order to functionally characterize the mutations they identified, the authors used a genome-wide genetic screening strategy to identify drug–gene interactions and determine why the relapse-specific mutational landscape is less responsive to chemotherapy. The screen was carried out in a representative model ALL cell line using CRISPR, a genetic editing tool used to specifically activate or inhibit the expression of single genes. 

Fig 1. Schematic of experimental design for CRISPR-based screen in ALL model cell line. A library of targeting molecules called guide RNAs (gRNAs) was used to activate or inhibit genes identified to have mutations associated with ALL in relapse. Figure adapted from Oshima, Zhao, Durán, Brown, et al. 2020.

Between these two approaches, the team succeeded in characterizing relapse-specific mutations that arise during the administration of chemotherapy itself, a process known as clonal evolution. Additionally, the number of mutations they identified increased with patient age at diagnosis, a finding that allowed the researchers to establish that the most recent commonality between the mutant cells present at diagnosis versus later at relapse often develops early, years before the leukemia is officially diagnosed. Importantly, this finding is consistent with the hypothesized fetal origin of many pediatric ALLs, which postulates that chromosomal abnormalities leading to cancer are already present at birth. It is also consistent with the higher rate of relapse previously observed in adult patients.

When the mutations uniquely acquired during relapse were further investigated using CRISPR screening in an ALL model cell line, a strong positive selection was revealed for those that conferred chemotherapy resistance. By using CRISPR to manipulate the expression of genes affected by each mutation of interest and assessing how the ALL model cells fared in each experiment, the researchers were able to analyze the relationships between the effect of the mutation and application of each drug. Of the drugs investigated, functional overlaps in the cellular mechanisms mediating the activity of each were identified between several groups of them. The significance of this finding is two-fold. First, it helps researchers and medical providers understand why the presently used multi-drug regimen might be effective for ALL in the first place. Second, it suggests that other drugs acting via similar mechanisms of action could be effective treatments in the future. Moving forward, ALL in relapse might be treated not just with combinatorial chemotherapy, but with specific combinations, doses, and schedules of drugs that meet the personalized genetic vulnerabilities of specific ALL cases. 

One inhibitor tested in the study’s cell-based CRISPR screen, an inhibitor called ABT-199, also known as Venetoclax, is already being tested for inclusion as a new therapeutic. If approved, it could become part of the arsenal of drugs used to compose personalized chemotherapeutic cocktails for patients with ALL in relapse. According to co-first author Dr. Brown, “it is currently in Phase I/II clinical trials for relapsed ALL and other malignancies and we hope that this work and our follow-up studies can further underscore the mechanisms of action of this inhibitor in combination with commonly used chemotherapies.” 

Altogether, this study identified a number of mutations that make relapsed ALL distinct from ALL before treatment with chemotherapy and functionally characterized the interactions of these mutations with multiple chemotherapy drugs. While ALL is not a common cancer – it accounts for less than 0.05% of all incidences of cancer in the United States – those affected must be treated with aggressive chemotherapy that can negatively affect the lives and health of patients in many ways. Because of this, understanding how to better target ALL at diagnosis and treatment-resistant ALL in relapse is a high priority for researchers. The findings of this work help to identify targets for reversing chemotherapy resistance and improving treatment outcomes for pediatric and adult patients alike. 

Dr. Jessie Brown is a postdoctoral research fellow in the Ferrando Lab at Columbia University Irving Medical Center studying therapeutic resistance in relapsed acute lymphoblastic leukemia.  

Musseling through climate change

Our planet’s climate is pretty old — an estimated 3.5 billion years old, in fact. Understanding how Earth’s climate has changed since then is important for predicting and coping with climate change today and in the future. But, because it is hard to know exactly what happened a couple of billion years ago, climate scientists use mathematically constructed models that take into account abiotic, or non-living, factors like carbon dioxide levels and ocean chemistry in the past to predict weather patterns in the future. Ultimately, the goal is to predict how biotic factors — living things like us — will be affected. 

These mathematical models are a work in progress. Often, they are made using data from field studies conducted over periods of only one to two years. Additionally, many models do not factor in biological mechanisms for plasticity that allow organisms to adapt to changing environmental conditions. These gaps were the impetus for a study conducted by Dr. Luca Telesca and colleagues, recently published in Global Change Biology. Their work investigated shell shape and body structure in archival specimens (read: preserved in ethanol, not fossilized) of the blue mussel (Mytilus edulis) collected roughly every decade between 1904 and 2016 along 15 kilometers of Belgium’s coast (Fig. 1). Measurements of the mussels themselves were coupled with extensive long-term datasets of coastline environmental conditions over the past century, all of which were obtained from collections at the Royal Belgian Institute of Natural Sciences

Fig. 1: Study location, along 15 km of Belgian coast between the cities of Ostend and Nieuwpoort (starred). Image source: Google Maps.

The blue mussel is not your typical specimen in an archival collection. Common animals often aren’t considered worth preserving for the historical record. However, it’s precisely because they are common that species like the blue mussel make great barometers for environments gone by. The blue mussel in particular is an example of a “calcifying foundation species,” species so named for their ability to sequester and store calcium and carbon from the surrounding water (see Fig. 2), and their habitat on shallow marine floors. This calcifying ability, or biomineralization, is the process by which living organisms convert non-living organic substances into still-non-living inorganic derivatives. It is an astoundingly ubiquitous process: all six taxonomic kingdoms from single-cell organisms in Archaea to mammals like us — we’re in the kingdom Animalia — contain organisms capable of biomineralization. The bones in our bodies are an example of this, the result of binding calcium phosphate from our diets into a different, crystallized form of calcium called hydroxyapatite. Furthermore, because biomineralization is an easy-to-measure, direct interaction between biotic and abiotic factors, it is an ideal study for climate scientists. 

Fig. 2: A typical blue mussel shell and cross-section. After calcium carbonate crystals are absorbed from the surrounding water, they become layered with secreted structural proteins from the mussel’s body tissue, or mantle. These layers of calcium carbonate and secreted proteins form the mussel’s shell, the thickness of which can vary depending on how much calcium carbonate is absorbed. Image created using Biorender.com.

One of the most pressing concerns presented by rapid climate change today is ocean acidification, characterized by an increase in oceanic carbonic acid resulting from elevated levels of carbon dioxide in the atmosphere. Excess carbonic acid increases the acidity of ocean water, which can dissolve shells, and decreases the availability of calcium carbonate, the nutrient that mussels and other ocean biomineralizers use to form shells in the first place. Ocean acidification has had a negative impact on many species; one notable impact is on coral in the Great Barrier Reef. Given these known effects of climate change and ocean acidification on many ocean calcifers, the authors predicted that they would observe a steady decrease in shell size between 1904 and 2016. 

Instead, to their surprise, they observed a marked increase in blue mussel shell size since 1904. The team’s results hold a number of implications for predictive climate change modeling. First, the findings signify that archival collections of organisms from the past can and should be used to influence our current predictions about what’s to come in any given biome, 10, 20, or 100 years from now. Second, and quite hopefully, the findings suggest that mussel populations somehow acclimated to shifting environmental conditions along the Belgian coast over the past century. The authors speculate that this could be because rising ocean temperatures could actually increase calcification, combating dissolution induced by acidic conditions, or that rising water temperatures may have increased the availability of a specific food source. Altogether, the potential for compensatory mechanisms in this study population of blue mussels points to the same potential in other species for coping with rapid environmental change over the next century. As we continue to update predictive models with data from the past and study and protect the populations most vulnerable to rapid climate change, we may find ways to help them mussel through yet. 

 

Dr. Telesca is a postdoctoral research scientist affiliated with Columbia University’s Earth Institute and the Lamont-Doherty Earth Observatory.