We explore how environmental factors impact human cell metabolism with relevance to hematological cancers and immunology
Cancer cells require metabolic adaptations to satisfy demands of proliferation and survival. Though this framework of metabolic rewiring is a nearly universal characteristic of cancer, the mechanisms and contexts that dictate various metabolic preferences and liabilities harbored by malignant cells remain poorly understood. Similar to malignant cells, normal lymphocytes must also adapt metabolism to satisfy proliferative demands upon their activation from a naïve state. In addition, emerging evidence suggests that transitions in the metabolic phenotype of different immune cells are also directly linked with differentiation and various effector functions. Taken together, interest to exploit altered cancer metabolism or to modulate immunometabolism for patient benefit will require an improved understanding of the metabolic regulation, preferences, and requirements of these diverse cell types. Given the scope and flexibility of experimentation possible through the study of cultured cells, in vitro models remain essential systems for addressing fundamental questions in human cell biology as well as for developing therapeutic pipelines.
However, while the influence of environmental factors on cellular metabolism has become increasingly appreciated, conventional models fail to recapitulate physiologic conditions and also limit efforts to interrogate distinct aspects of the environment in isolation.
Our lab is interested in combining cutting-edge technologies with novel tools that we develop to better understand how environmental factors influence the metabolic landscape of diverse hematological cancers and normal lymphocytes. We apply a highly interdisciplinary approach that combines principles from biochemistry, engineering, and molecular biology with methods in metabolomics, functional genomics, and chemical genetics.
Our goal is to discover and characterize unforeseen biological (and pharmacological) phenomena that may have been missed or misinterpreted owing to the use of traditional in vitro (and perhaps even in vivo) model systems that poorly mimic physiologic conditions, and therefore, that inadequately capture the contribution of environmental factors to shaping cell metabolism. Ultimately, we hope to translate these findings into new therapeutic opportunities (e.g. small molecule targeted therapy or direct manipulation of environmental conditions) for cancer intervention.
- Tool Development
In previous work, we systematically developed a new cell culture medium (human plasma-like medium; HPLM) that contains a defined collection of > 50 polar metabolites and salt ions at concentrations that reflect the average values of those reported for normal adult human plasma. We then demonstrated that, relative to culture in an established medium, HPLM induced widespread effects across the metabolic landscape of various cell types. Through the use of HPLM, we also discovered that uric acid, at concentrations present in human but not mouse plasma (nor established media), directly inhibits the enzyme UMP synthase (UMPS), which in turn, reduces cellular sensitivity to the classic chemotherapeutic 5-fluorouracil (5-FU) (Cantor et al., Cell, 2017).
Ultimately, this story highlighted that HPLM could be exploited to uncover an example of metabolic regulation that would have been difficult to discover otherwise using existing model systems, whether that be studying cells cultured in established media or grown in mice. More generally, this study revealed the impact of media composition on cellular metabolism, suggesting that HPLM should be of broad utility to study both biological and pharmacological questions in a more physiologically relevant context.
We are now interested in developing additional HPLM derivatives that expand upon our initial formulation, or that reflect the environmental conditions associated with different pathophysiological states or sites within the body. Indeed, relative to our initial published recipe, we have already been utilizing in our current studies a basal HPLM that contains 4 additional components.
Interested in trying HPLM?
While we continue progress toward making HPLM commercially available, please contact Jason to inquire about receiving a sample of the homemade formulation.
We are also interested in utilizing in vitro systems that complement conventional cell culture methods. In particular, drawing from concepts in bioreactor design typically associated with chemical engineering, we previously adapted mammalian cell bioreactors to operate as chemostats. In contrast to typical (batch) cell culture, chemostats are continuous flow systems that enable cell growth under tightly controlled and constant conditions, i.e. operation at steady state. A primary advantage of steady state cell culture is the potential to study phenotypic consequences of altering a single environmental parameter while keeping all others constant, thus limiting secondary effects.
(Barcoded) Human blood cancer cell line collection
Cancer is a remarkably complex collection of diseases, which makes it unsurprising that different cancer (sub)types are marked by heterogeneous metabolic preferences and dependencies, therapeutic regimens, and treatment outcomes (Cantor and Sabatini, Cancer Discov., 2012). Given our interest in studying hematological cancers, we previously constructed a collection of over 50 human blood cancer cell lines that together sample various subtypes of leukemia, lymphoma, and myeloma. Across nearly our entire collection, we have also engineered individual cell lines to harbor unique DNA barcodes, which in turn, permits several cell lines to be cultured and tracked in a pooled fashion. Such an approach was described previously by others (Birsoy et al., Nature, 2014), and an iteration of our barcoded blood cancer cell line collection was used analogously in a recent study (Kanarek et al., Nature, 2018).
- Research Areas
Relative to traditional media, our use of a cell culture medium (HPLM) that better recapitulates the metabolic composition of human plasma induced widespread effects on the metabolism of cultured cells. Among a panel of diverse blood cancer cell lines, such alterations could be categorized as either shared or cell line-specific, underscoring how the heterogeneity of cancer can influence cell responses to the same environmental conditions. Using biochemistry and various approaches in metabolomics, we aim to further explore and characterize the physiologic functions of exogenous metabolites and to understand how the metabolic activities across diverse human blood cancers are affected by environmental factors that better recapitulate a physiological context.
Large-scale CRISPR-based screens make it possible to identify genes required for the survival and proliferation of mammalian cells, and therefore, have offered a promising approach to better understand gene function and to identify therapeutic targets in human disease. However, despite a growing appreciation that gene essentiality is not exclusively a cell-intrinsic property, such screens have relied on the interrogation of cells cultured in established media that poorly reflect the metabolic composition of human blood. Using a comparative functional genomics approach, we propose to identify genetic deletions that differentially affect cell fitness in HPLM relative to traditional media. We will then leverage methods in traditional biochemistry and metabolomics to determine why particular genetic deletions impart such differential effects on cell growth, as well as to characterize and understand the functional role(s) of proteins encoded by these genes of interest. Ultimately, we hope that these approaches will reveal unforeseen therapeutic opportunities with greater in vivo relevance.
Although cell lines remain integral to therapeutic discovery and development efforts, there is a well-recognized disconnect between the pre-clinical and clinical success of many drug candidates. This significant problem has been largely attributed to the inability of existing in vitro models to mimic a relevant environmental context, with most such ensuing consideration given to differential effects induced by 2D and 3D culture systems, but little to the non-physiologic metabolite composition of traditional media. We propose to use a comparative chemical genetics approach to more broadly evaluate chemical perturbations that differentially affect cell viability in HPLM relative to traditional media. Through an ongoing collaboration with Dr. Matthew Hall at NIH/NCATS, we utilize a library of over 1,900 oncology-focused small molecule compounds to carry out initial quantitative high-throughput screens. Guided by such screens, we aim to understand the underlying mechanism(s) that dictate medium-mediated differential drug phenotypes. Ultimately, we hope to identify more physiologically relevant drug phenotypes, and in turn, to uncover novel metabolite-drug interactions and the corresponding metabolic pathways that contribute to drug potency.
Blood cancer nutrient dependencies
Several cancer types are auxotrophic for one or more amino acids and therefore rely on import of a particular amino acid(s) from the serum to survive. This type of liability can be exploited through systemic depletion of the circulating “cancer-essential” amino acid (Cantor et al., Methods Enzymol., 2012). The most notable example of this strategy is the clinucak success of asparaginase in treating acute lymphoblastic leukemia (ALL). Whereas normal cells can produce the non-essential amino acid asparagine (L-Asn) de novo via asparagine synthetase (ASNS), certain ALL cells express very low levels of ASNS and require uptake of serum L-Asn to survive. Asparaginase depletes serum L-Asn, resulting in selective induction of apoptosis in the auxotrophic ALL cells. However, ASNS levels do not provide an absolute predictive indication of susceptibility to asparaginase treatment, strongly indicating that unbiased approaches could more effectively identify the heterogeneous dependencies of blood cancers to different exogenous biomolecules. We will first utilize our unique set of tools to identify such liabilities, and then combining approaches ranging from expression analyses to metabolomics and CRISPR-based screens, we will aim to determine why certain cells are sensitive to the absence of a particular nutrient, whereby such phenotypes may be indeed dependent upon culture in a more physiologically relevant medium.
There has been a recent surge of interest in understanding how the metabolic phenotypes of different immune cells are directly linked to cellular differentiation state and effector functions. Within this space, there has been a growing appreciation that environmental factors can have dramatic effect on immune cell function, with a particular focus on (altered) availability or accumulation across a small number of exogenous nutrients. Further, like our understanding of cancer cell metabolism, that of normal lymphocytes and other immune cells is largely based on in vitro experimentation using cell lines or cells isolated from tumors or blood. Combining the tools that we have developed with methods in both metabolomics and immunobiology, we are interested in examining how the metabolic phenotypes of normal lymphocytes are affected by environmental factors that more closely reflect physiological contexts. We aim to better characterize and understand the functional relationship between exogenous metabolites and lymphocyte metabolism, and ultimately, we hope to translate these findings into new approaches in modulating immune cell function.