Deconstructing cancer with precision genome editing.

Recent advances in genome editing technologies are allowing investigators to engineer and study cancer-associated mutations in their endogenous genetic contexts with high precision and efficiency. Of these, base editing and prime editing are quickly becoming gold-standards in the field due to their versatility and scalability. Here, we review the merits and limitations of these precision genome editing technologies, their application to modern cancer research, and speculate how these could be integrated to address future directions in the field.

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Mechanisms of Resistance to Chimeric Antigen Receptor T Cell Therapy.

CAR T cell therapy has significantly shaped the treatment landscape for refractory hematologic malignancies including large B-cell lymphomas, multiple myeloma, and leukemias. While response rates for a previously dismal prognosis have improved, certain obstacles still remain to achieving CAR T infallibility. In this article, we review the data surrounding proposed resistance mechanisms of tumors to CAR T, including the implications of target loss, exhausted T cells as effete effectors, the necessity of maximal CAR T expansion to durable response, the negative impact of an inflammatory milieu and a suppressive tumor microenvironment, and the optimal tumor-to-effector ratio that associates with best outcomes. The future of CAR T should aim to mitigate these weaknesses in order to bolster the efficacy of this revolutionary therapy.

A Case of Panic Attacks Developing After 10 Years of Chronic Cannabis Use in a Patient With No Prior Psychiatric History.

Cannabis use for medical and recreational purposes is increasing. Inhibitory activity of cannabinoids (CB) at the CB1 and CB2 receptors centrally and peripherally mediate the therapeutic effects that are wielded for palliation of pain, anxiety, inflammation, and nausea in indicated conditions. Cannabis dependence is also associated with anxiety; however, the direction of causality is unknown, such as whether anxiety disorders lead to cannabis use, or whether cannabis contributes to the development of anxiety disorder. The evidence hints that both may have validity. Here we present a case of cannabis-associated panic attacks following 10 years of chronic cannabis dependence in an individual with no prior psychiatric history. The patient is a 32-year-old male with no significant past medical history who presented complaining of five-minute episodes of palpitations, dyspnea, upper extremity paresthesia, subjective tachycardia, and cold diaphoresis occurring in a variety of circumstances for the past two years. His social history was significant for 10 years of smoking marijuana multiple times daily, which he had quit over two years ago. The patient denied past psychiatric history or known anxiety problems. Symptoms were unrelated to activity and only relieved with deep breathing. The episodes were not associated with chest pain, syncope, headache, or emotional triggers. The patient had no family history of cardiac disease or sudden death. The episodes were refractory to the elimination of caffeine, alcohol, or other sugary beverages. The patient had already stopped smoking marijuana when the episodes began. Due to the unpredictable nature of the episodes, the patient reported a growing fear of being in public. On laboratory workup, metabolic and blood panels were within normal limits, as well as thyroid studies. Electrocardiogram showed normal sinus rhythm, and continuous cardiac monitoring revealed no arrhythmias or abnormalities despite the patient indicating multiple triggered events within the duration of monitoring. Echocardiography also showed no abnormalities. With organic cardiac causes of the subjective palpitation episodes ruled out, a psychogenic etiology of the episodes was presumed, and the patient was referred to behavioral health services. In conclusion, cannabis-induced anxiety or panic disorders should be considered in patients with no prior psychiatric history presenting with anxiety-like attacks following a period of cannabis dependence or current use. These patients should be advised to cease cannabis use and referred to behavioral medicine.

Two MYB Proteins in a Self-Organizing Activator-Inhibitor System Produce Spotted Pigmentation Patterns.

Many organisms exhibit visually striking spotted or striped pigmentation patterns. Developmental models predict that such spatial patterns can form when a local autocatalytic feedback loop and a long-range inhibitory feedback loop interact. At its simplest, this self-organizing network only requires one self-activating activator that also activates a repressor, which inhibits the activator and diffuses to neighboring cells. However, the molecular activators and inhibitors fully fitting this versatile model remain elusive in pigmentation systems. Here, we characterize an R2R3-MYB activator and an R3-MYB repressor in monkeyflowers (Mimulus). Through experimental perturbation and mathematical modeling, we demonstrate that the properties of these two proteins correspond to an activator-inhibitor pair in a two-component, reaction-diffusion system, explaining the formation of dispersed anthocyanin spots in monkeyflower petals. Notably, disrupting this pattern impacts pollinator visitation. Thus, subtle changes in simple activator-inhibitor systems are likely essential contributors to the evolution of the remarkable diversity of pigmentation patterns in flowers.