5G2 mutant mice model loss of a commonly deleted segment of chromosome 7q22 in myeloid malignancies.

Monosomy 7 and del(7q) are among the most common and poorly understood genetic alterations in myelodysplastic neoplasms and acute myeloid leukemia. Chromosome band 7q22 is a minimally deleted segment in myeloid malignancies with a del(7q). However, the rarity of "second hit" mutations supports the idea that del(7q22) represents a contiguous gene syndrome. We generated mice harboring a 1.5 Mb germline deletion of chromosome band 5G2 syntenic to human 7q22 that removes Cux1 and 27 additional genes. Hematopoiesis is perturbed in 5G2+/del mice but they do not spontaneously develop hematologic disease. Whereas alkylator exposure modestly accelerated tumor development, the 5G2 deletion did not cooperate with KrasG12D, NrasG12D, or the MOL4070LTR retrovirus in leukemogenesis. 5G2+/del mice are a novel platform for interrogating the role of hemopoietic stem cell attrition/stress, cooperating mutations, genotoxins, and inflammation in myeloid malignancies characterized by monosomy 7/del(7q).

KrasP34R and KrasT58I mutations induce distinct RASopathy phenotypes in mice.

Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.

An In Vivo Kras Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants.

KRAS is the most frequently mutated oncogene in cancer, yet there is little understanding of how specific KRAS amino acid changes affect tumor initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRASG12C), pancreas (KRASG12R), and colon (KRASG13D) cancers. Induction of each allele in either the murine colon or pancreas revealed striking quantitative and qualitative differences between KRAS mutants in driving the early stages of transformation. Furthermore, using pancreatic organoid models, we show that KRASG13D mutants are sensitive to EGFR inhibition, whereas KRASG12C-mutant organoids are selectively responsive to covalent G12C inhibitors only when EGFR is suppressed. Together, these new mouse strains provide an ideal platform for investigating KRAS biology in vivo and for developing preclinical precision oncology models of KRAS-mutant pancreas, colon, and lung cancers. SIGNIFICANCE: KRAS is the most frequently mutated oncogene. Here, we describe new preclinical models that mimic tissue-selective KRAS mutations and show that each mutation has distinct cellular consequences in vivo and carries differential sensitivity to targeted therapeutic agents.See related commentary by Kostyrko and Sweet-Cordero, p. 1626.This article is highlighted in the In This Issue feature, p. 1611.

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model.

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.

Prolongation and enhancement of the anti-apoptotic effects of PTD-Hsp27 fusion proteins using an injectable thermo-reversible gel in a rat myocardial infarction model.

Ischemic heart disease has emerged as a leading cause of death worldwide. Conventional surgery-based therapy for this disease, especially myocardial infarction, requires additional pharmaceutical agents using heart's endogenous protective mechanism to suppress the progress and recurrence of the disease. Heat shock protein 27 (Hsp27) has been considered to be a potentially therapeutic protein for the treatment of ischemic heart disease due to its anti-apoptotic and protective effects on cardiomyocytes under stressful conditions. Despite the potency of Hsp27, low transduction efficiency, protein instability, and a short half-life in the body have limited its in vivo applications. Protein transduction domains (PTD) were recombinantly fused with Hsp27 to enhance transduction efficiency. Although the intracellular delivery of the PTD-Hsp27 fusion proteins was significantly enhanced compared with Hsp27, the instability and short half-life of the PTD-Hsp27 fusion proteins still need to be improved for in vivo applications. Injectable thermo-reversible gel system was developed and found to be effective in stabilizing and retarding the release of the PTD-Hsp27 fusion proteins both in vitro and in vivo. PTD-Hsp27-loaded thermo-reversible gels were locally administered to the heart muscle in a ligation/reperfused rat myocardial infarction model and the long-term therapeutic efficacy was observed by measuring the inhibition of apoptosis and the area of fibrosis.