Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes.

Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes.

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are aggressive myeloproliferative neoplasms that are incurable with conventional chemotherapy. Mutations that deregulate Ras signaling play a central pathogenic role in both disorders, and Mx1-Cre, KrasLSL-G12D mice that express the Kras oncogene develop a fatal disease that closely mimics these two leukemias in humans. Activated Ras controls multiple downstream effectors, but the specific pathways that mediate the leukemogenic effects of hyperactive Ras are unknown. We used PD0325901, a highly selective pharmacological inhibitor of mitogen-activated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK signaling drives neoplasm formation in Mx1-Cre, KrasLSL-G12D mice. PD0325901 treatment induced a rapid and sustained reduction in leukocyte counts, enhanced erythropoiesis, prolonged mouse survival, and corrected the aberrant proliferation and differentiation of bone marrow progenitor cells. These responses were due to direct effects of PD0325901 on Kras mutant cells rather than to stimulation of normal hematopoietic cell proliferation. Consistent with the in vivo response, inhibition of MEK reversed the cytokine hypersensitivity characteristic of KrasG12D hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasias, and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.

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Germ line missense mutations in HRAS and KRAS and in genes encoding molecules that function up-or downstream of Ras in cellular signaling networks cause a group of related developmental disorders that includes Costello syndrome, Noonan syndrome, and cardiofaciocutaneous syndrome. We performed detailed biochemical and functional studies of three mutant K-Ras proteins (P34R, D153V, and F156L) found in individuals with Noonan syndrome and cardiofaciocutaneous syndrome. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase-activating proteins, and guanine nucleotide dissociation all regulate developmental programs in vivo.

Based on clinical presentation, glioblastoma (GBM) is stratified into primary and secondary types. The protein 53 (p53) pathway is functionally incapacitated in most GBMs by distinctive type-specific mechanisms. To model human gliomagenesis, we used a GFAPHRas V12 mouse model crossed into the p53ER TAM background, such that either one or both copies of endogenous p53 is replaced by a conditional p53ER TAM allele. The p53ER TAM protein can be toggled reversibly in vivo between wild-type and inactive conformations by administration or withdrawal of 4-hydroxytamoxifen (4-OHT), respectively. Surprisingly, gliomas that develop in GFAP-HRas V12 ; p53 +/KI mice abrogate the p53 pathway by mutating p19 ARF / MDM2 while retaining wild-type p53 allele. Consequently, such tumors are unaffected by restoration of their p53ER TAM allele. By contrast, gliomas arising in GFAP-HRas V12 ;p53 KI/KI mice develop in the absence of functional p53. Such tumors retain a functional p19 ARF /MDM2-signaling pathway, and restoration of p53ER TAM allele triggers p53-tumor-suppressor activity. Congruently, growth inhibition upon normalization of mutant p53 by a small molecule, Prima-1, in human GBM cultures also requires p14 ARF /MDM2 functionality. Notably, the antitumoral efficacy of p53 restoration in tumor-bearing GFAP-HRas V12 ;p53 KI/KI animals depends on the duration and frequency of p53 restoration. Thus, intermittent exposure to p53ER TAM activity mitigated the selective pressure to inactivate the p19 ARF /MDM2/p53 pathway as a means of resistance, extending progression-free survival. Our results suggest that intermittent dosing regimes of drugs that restore wild-type tumorsuppressor function onto mutant, inactive p53 proteins will prove to be more efficacious than traditional chronic dosing by similarly reducing adaptive resistance.preclinical model | Nutlin 3 | intermittent treatment

IntroductionZika virus, an arbovirus of the Flaviviridae family, is a mosquito-borne virus known to cause microcephaly through vertical transmission. Infection presents with mild, self-limiting symptoms. Currently, a Zika virus outbreak has spread across most of South and Central America. Travel-related and sexually transmitted cases have been reported across the United States. However, the vector-borne transmission has been limited to Florida and Texas. We present seven cases of Zika virus infection that presented at a single institution in South Florida.MethodsPatients were included that had real-time polymerase-chain reaction (RT-PCR) for Zika virus RNA in urine or serum or enzyme-linked immunosorbent assay (ELISA) for Immunoglobulin M (IgM) antibody against Zika virus in serum.ResultsAll seven patients reported recent travel or employment in areas of active Zika virus transmission and at least two of the four most commonly reported symptoms (fever, arthralgia, rash, and conjunctivitis) with a rash present in all patients. All patients had positive RT-PCR for Zika virus RNA in urine. RT-PCR for Zika virus RNA in serum was negative in four of five patients that were tested, indicating that these patients likely presented one to two weeks after symptom onset.ConclusionThe future of Zika virus outbreaks in other cities in the United States is still uncertain. However, it is clear that prevention and control policies are urgently needed. We have presented seven confirmed cases of Zika virus infection in South Florida. In addition to conducting research concerning both the diagnostic and therapeutic aspects of the virus, there is a need for public awareness of its presentation, methods of transmission, and subsequent clinical outcomes.

966 Mutations that deregulate cellular signaling are a hallmark of myeloproliferative neoplasms (MPNs), and pharmacologic inhibitors of MPN-associated proteins have redefined therapy for some MPNs. However, this strategy cannot yet be applied to juvenile- and chronic myelomonocytic leukemias (JMML and CMML). These diseases are characterized by aberrant N-Ras, K-Ras, Cbl, and SHP-2 proteins that are not easily targeted by drugs. An attractive alternative approach is to inhibit downstream effector pathways, which include the Raf/MEK/ERK, phosphoinositide-3-OH kinase (PI3K)/Akt, and Ral-GDS/Ral-A cascades. However, it is not known which of these pathways are crucial for the aberrant growth and survival of JMML and CMML cells and might therefore provide the best targets for therapy. To address these questions, we developed an accurate mouse model of JMML and CMML by expressing a conditional “knock-in” KrasLSL-G12D oncogene in bone marrow. We administered PD0325901, a potent and selective MEK inhibitor, to Mx1-Cre, KrasG12D mutant mice to test the hypothesis that the Raf→MEK→ERK cascade is necessary for MPN initiated by KrasG12D expression. Oral administration of PD0325901 5 mg/kg caused deep and durable MEK inhibition in primary bone marrow progenitors. Mx1-Cre, KrasG12D mice with established MPN and wild-type (WT) littermates were randomly assigned to receive PD0325901 5 mg/kg/day or a control vehicle. Treated Mx1-Cre, KrasG12D mice demonstrated rapid correction of leukocytosis and anemia, and reduction in splenomegaly. Treatment was also associated with dramatic improvement in the survival of Mx1-Cre, KrasG12D mice (8.1 vs. 2.0 weeks after entry, p=0.003). Two of three Mx1-Cre, KrasG12D mice that were treated for 12 weeks ultimately died with KrasG12D T-lineage leukemia/lymphoma, but none succumbed with progressive MPN. Flow cytometry of bone marrow and peripheral populations showed that PD0325901 reversed the granulocyte/monocyte progenitor bias and ineffective erythropoiesis in KrasG12D mice. However, PD0325901 did not eliminate the rearranged mutant Kras allele in myeloid progenitors, and these cells remained hypersensitive to GM-CSF in methylcellulose cultures. Therefore, PD0325901 did not eliminate Kras mutant cells, but rather modified their behavior in vivo so as to restore a normal output of the hematopoietic system. To further address the biologic effects of PD0325901 on growth of primary progenitor cells in vitro, we examined colony growth over a range of GM-CSF concentrations. Importantly, whereas in vitro exposure to PD0325901 did not selectively abrogate colony growth from bone marrow of naïve Mx1-Cre, KrasG12D mice in the presence of saturating doses of GM-CSF, a low concentration of PD0325901 eliminated the growth of cytokine-independent progenitor colonies. Even more strikingly, this also restored a normal GM-CSF dose response curve in clonogenic progenitors, eliminating the hypersensitive growth pattern that is a hallmark of MPN. Finally, even at saturating doses of GM-CSF, a low concentration of PD0325901 was sufficient to normalize the numbers and types of cells within the colonies. Together, these data show that a low concentration of PD0325901 is sufficient to impart a normal program of proliferation and differentiation in KrasG12D myeloid progenitors. These findings are highly consistent with the in vivo data. Collectively, our data suggest that aberrant MEK activation mediates most aspects of the MPN phenotype in the progenitor compartment and support the development of clinical trials to evaluate MEK inhibitors in patients with JMML and CMML. Disclosures: No relevant conflicts of interest to declare.