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The combination of the synthetic TLR9 ligand CpG and agnostic OX40 antibody can trigger systemic antitumor immune responses upon co-injection into the tumor microenvironment, eradicating simultaneous untreated sites of metastatic disease. Here we explore the application of this in situ immunotherapy to the neoadjuvant setting. Current neoadjuvant checkpoint blockade therapy is delivered systemically, resulting in off-target adverse effects. In contrast, intratumoral immunotherapy minimizes the potential for toxicities and allows for greater development of combination therapies. In two metastatic solid tumor models, neoadjuvant intratumoral immunotherapy generated a local T-cell antitumor response that then acted systemically to attack cancer throughout the body. In addition, the importance of timing between neoadjuvant immunotherapy and surgical resection was established, as well as the increased therapeutic power of adding systemic anti-PD1 antibody. The combination of local and systemic immunotherapy generated an additional survival benefit due to synergistic inhibitory effect on tumor-associated macrophages. These results provide a strong rationale for translating this neoadjuvant intratumoral immunotherapy to the clinical setting, especially in conjunction with established checkpoint inhibitors.Significance:This work demonstrates the ability of neoadjuvant intratumoral immunotherapy to target local and distant metastatic disease and consequently improve survival.

The combination of the synthetic TLR9 ligand CpG and agnostic OX40 antibody can trigger systemic antitumor immune responses upon co-injection into the tumor microenvironment, eradicating simultaneous untreated sites of metastatic disease. Here we explore the application of this in situ immunotherapy to the neoadjuvant setting. Current neoadjuvant checkpoint blockade therapy is delivered systemically, resulting in off-target adverse effects. In contrast, intratumoral immunotherapy minimizes the potential for toxicities and allows for greater development of combination therapies. In two metastatic solid tumor models, neoadjuvant intratumoral immunotherapy generated a local T-cell antitumor response that then acted systemically to attack cancer throughout the body. In addition, the importance of timing between neoadjuvant immunotherapy and surgical resection was established, as well as the increased therapeutic power of adding systemic anti-PD1 antibody. The combination of local and systemic immunotherapy generated an additional survival benefit due to synergistic inhibitory effect on tumor-associated macrophages. These results provide a strong rationale for translating this neoadjuvant intratumoral immunotherapy to the clinical setting, especially in conjunction with established checkpoint inhibitors. Significance: This work demonstrates the ability of neoadjuvant intratumoral immunotherapy to target local and distant metastatic disease and consequently improve survival.

The unprecedented benefits of immunotherapy in advanced malignancies have resulted in increased interests in exploiting immune stimulatory agents in earlier-stage solid tumors in the neoadjuvant setting. However, systemic delivery of immunotherapies may cause severe immune-related side-effects and hamper the development of combination treatments. Intratumoral delivery of neoadjuvant immunotherapy provides a promising strategy in harnessing the power of immunotherapy while minimizing off-target toxicities. The direct injection of immune stimulating agents into the tumor primes the local tumor-specific immunity to generate a systemic, durable clinical response. Intratumoral immunotherapy is a highly active area of investigation resulting in a plethora of agents, for example, immune receptor agonists, non-oncolytic and oncolytic viral therapies, being tested in preclinical and clinical settings. Currently, more than 20 neoadjuvant clinical trials exploring distinct intratumoral immune stimulatory agents and their combinations are ongoing. Practical considerations, including appropriate timing and optimal local delivery of immune stimulatory agents play an important role in safety and efficacy of this approach. Here, we discuss promising approaches in drug delivery technologies and opportunity for combining intratumoral immunotherapy with other cancer treatments and summarize the recent preclinical and clinical evidences that highlighted its promise as a part of routine oncologic care.

Objective The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. Summary of background data Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. Methods To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. Results Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. Conclusion Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

Objective: Fetuses early in gestation heal skin wounds without forming scars. The biological mechanisms behind this process are largely unknown. Fibroblasts, however, are cells known to be intimately involved in wound healing and scar formation. We examined fibroblasts in different stages of development to characterize differences in gene expression that may result in the switch from regenerative wound repair to repair with scarring.

Background Cancer surgery remains the most effective single treatment modality for curing patients with solid tumors. However, only half of all cancer patients undergo curative resection. Of those, a significant number will eventually relapse from occult metastases. Administration of therapy prior to surgery, known as neoadjuvant therapy, has revolutionized cancer surgery. It not only decreases overall tumor burden, rendering previously inoperable disease operable, but also improves survival by providing systemic disease control in patients at high risk of having microscopic distant metastases at the time of surgery. While traditional neoadjuvant therapy using conventional chemotherapy and radiation therapy has improved patient outcomes across a spectrum of solid tumors, the potential of neoadjuvant immunotherapy is currently unknown. Various immune modulating agents have achieved significant clinical success in the management of metastatic and treatment resistant cancers, yet their utility in the neoadjuvant setting remains unclear. In theory, neoadjuvant immunotherapy is particularly interesting because it utilizes the primary tumor as a rich source of antigens for T cell priming. As a result, it may be successful in eliciting an immune response where adjuvant immunotherapy treatment has failed. Here we designed a preclinical study utilizing neoadjuvant immunotherapy and demonstrate the efficacy of employing immune stimulating agents synergistically with surgical resection. Methods We used two different murine models of solid tumors – a metastatic triple negative breast carcinoma and a colorectal carcinoma to compare neoadjuvant immunotherapy, resection alone, or immunotherapy alone treatments on local and systemic disease control, and survival. We found that neoadjuvant immunotherapy offered the best survival as well as the best locoregional and metastatic disease control when compared to the other groups. In fact, in the highly aggressive and spontaneously metastatic breast carcinoma model the neoadjuvant treatment group was the only group in which a long-term surviving cohort of mice was cured. When these long-term survivors were rechallenged with the same tumor, they were immune, confirming that the treatment successfully conferred immunological memory. When exposed to a new tumor type, however, they remained susceptible, confirming the specificity of this immunologic memory. Disease control was found to be T cell dependent as depletion of either both CD4+ and CD8+, or CD8+ only T cells resulted in loss of therapeutic benefit in mice treated with neoadjuvant immunotherapy. Conclusion We found that neoadjuvant immunotherapy successfully initiated cell mediated antitumor activity both locally and systemically at an anatomically distant disease site. This resulted in improved survival in the neoadjuvant group when compared to groups receiving resection alone or immunotherapy alone. As increased options to incorporate immunotherapy in the clinic become available to clinicians, this study explores the timely and clinically important question of how immunotherapy may optimally be combined with surgical resection. The results of the study are readily translatable and may be utilized to inform establishment of neoadjuvant immunotherapy regimens for patients with early stage and locally advanced solid tumors. Citation Format: Wan Xing Hong, Idit Sagiv-Barfi, Debra Czerwinski, Ronald Levy. Neoadjuvant immunotherapy for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5568.

Objective: In early gestation, fetal wounds heal without fibrosis in a process resembling regeneration. Elucidating this remarkable mechanism can result in tremendous benefits to prevent scarring. Fetal mouse cutaneous wounds before embryonic day (E)18 heal without scar. Herein, we analyze expression profiles of fetal and postnatal wounds utilizing updated gene annotations and pathway analysis to further delineate between repair and regeneration.

Fetal cutaneous wounds have the unique ability to completely regenerate wounded skin and heal without scarring. However, adult cutaneous wounds heal via a fibroproliferative response which results in the formation of a scar. Understanding the mechanism(s) of scarless wound healing leads to enormous clinical potential in facilitating an environment conducive to scarless healing in adult cutaneous wounds. This article reviews the embryonic development of the skin and outlines the structural and functional differences in adult and fetal wound healing phenotypes. A review of current developments made towards applying this clinical knowledge to promote scarless healing in adult wounds is addressed.

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen–dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.

The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

BackgroundHuman-induced pluripotent stem cells (iPSCs) are a potentially unlimited source for generation of cardiomyocytes (iPSC-CMs). However, current protocols for iPSC-CM derivation face several challenges, including variability in somatic cell sources and inconsistencies in cardiac differentiation efficiency.ObjectivesThis study aimed to assess the effect of epigenetic memory on differentiation and function of iPSC-CMs generated from somatic cell sources of cardiac versus noncardiac origins.MethodsCardiac progenitor cells (CPCs) and skin fibroblasts from the same donors were reprogrammed into iPSCs and differentiated into iPSC-CMs via embryoid body and monolayer-based differentiation protocols.ResultsDifferentiation efficiency was found to be higher in CPC-derived iPSC-CMs (CPC-iPSC-CMs) than in fibroblast-derived iPSC-CMs (Fib-iPSC-CMs). Gene expression analysis during cardiac differentiation demonstrated up-regulation of cardiac transcription factors in CPC-iPSC-CMs, including NKX2-5, MESP1, ISL1, HAND2, MYOCD, MEF2C, and GATA4. Epigenetic assessment revealed higher methylation in the promoter region of NKX2-5 in Fib-iPSC-CMs compared with CPC-iPSC-CMs. Epigenetic differences were found to dissipate with increased cell passaging, and a battery of in vitro assays revealed no significant differences in their morphological and electrophysiological properties at early passage. Finally, cell delivery into a small animal myocardial infarction model indicated that CPC-iPSC-CMs and Fib-iPSC-CMs possess comparable therapeutic capabilities in improving functional recovery in vivo.ConclusionsThis is the first study to compare differentiation of iPSC-CMs from human CPCs versus human fibroblasts from the same donors. The authors demonstrate that although epigenetic memory improves differentiation efficiency of cardiac versus noncardiac somatic cell sources in vitro, it does not contribute to improved functional outcome in vivo.

Significance: Poor wound healing remains a significant health issue for a large number of patients in the United States. The physiologic response to local wound hypoxia plays a critical role in determining the success of the normal healing process. Hypoxia-inducible factor-1 (HIF-1), as the master regulator of oxygen homeostasis, is an important determinant of healing outcomes. HIF-1 contributes to all stages of wound healing through its role in cell migration, cell survival under hypoxic conditions, cell division, growth factor release, and matrix synthesis throughout the healing process. Recent Advances: Positive regulators of HIF-1, such as prolyl-4-hydroxylase inhibitors, have been shown to be beneficial in enhancing diabetic ischemic wound closure and are currently undergoing clinical trials for treatment of several human-ischemia-based conditions. Critical Issues: HIF-1 deficiency and subsequent failure to respond to hypoxic stimuli leads to chronic hypoxia, which has been shown to contribute to the formation of nonhealing ulcers. In contrast, overexpression of HIF-1 has been implicated in fibrotic disease through its role in increasing myofibroblast differentiation leading to excessive matrix production and deposition. Both positive and negative regulators of HIF-1 therefore provide important therapeutic targets that can be used to manipulate HIF-1 expression where an excess or deficiency in HIF-1 is known to correlate with pathogenesis. Future Directions: Targeting HIF-1 during wound healing has many important clinical implications for tissue repair. Counteracting the detrimental effects of excessive or deficient HIF-1 signaling by modulating HIF-1 expression may improve future management of poorly healing wounds.

Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies.

ObjectivesThe purpose of this study was to evaluate whether radiation exposure from cardiac computed tomographic angiography (CTA) is associated with deoxyribonucleic acid (DNA) damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair.BackgroundExposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear.MethodsWe conducted a prospective cohort study in 67 patients undergoing cardiac CTA between January 2012 and December 2013 in 2 U.S. medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction.ResultsThe median dose length product was 1,535.3 mGy·cm (969.7 to 2,674.0 mGy·cm). The median radiation dose to the blood was 29.8 mSv (18.8 to 48.8 mSv). Median DNA damage increased 3.39% (1.29% to 8.04%, p < 0.0001) and median apoptosis increased 3.1-fold (interquartile range [IQR]: 1.4- to 5.1-fold, p < 0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 (1.9-fold [IQR: 1.5- to 3.0-fold], p < 0.001), XRCC4 (3.0-fold [IQR: 1.1- to 5.4-fold], p = 0.005), and BAX (1.6-fold [IQR: 0.9- to 2.6-fold], p < 0.001). Exposure to radiation was associated with DNA damage (odds ratio [OR]: 1.8 [1.2 to 2.6], p = 0.003). DNA damage was associated with apoptosis (OR: 1.9 [1.2 to 5.1], p < 0.0001) and gene activation (OR: 2.8 [1.2 to 6.2], p = 0.002).ConclusionsPatients exposed to >7.5 mSv of radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis--the keratinocyte and fibroblast--during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with2-fold expression differences with a false discovery rate2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

Human embryonic germ cells (hEGCs) are a valuable and underutilized source of pluripotent stem cells. Unlike embryonic stem cells, which have been extensively studied, little is known about the factors that regulate hEGC derivation and maintenance. This study demonstrates for the first time a central role for selective activation of PDGFR signaling in the derivation and maintenance of pluripotency in hEGCs. In the study, hEGCs were found to express PDGF receptor α at high levels compared to human embryonic stem cells (hESCs). PDGF significantly improved formation of alkaline phosphatase (AP) positive hEGC colonies. We subsequently determined that PDGF activates the phosphatidylinositol-3-kinase (PI3K) pathway as phosphorylation of AKT was up-regulated in response to PDGF. Furthermore, inhibition of PI3K signaling using small molecular inhibitor LY294002 led to significantly decreased AP positive hEGC colony formation whereas inhibition of MAPK pathway using U0126 had a negligible effect. We established a primary mechanism for PDGF mediated derivation and maintenance of hEGCs by demonstrating that OCT4 was upregulated and PTEN was suppressed in a dose dependent manner in response to PDGF.

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, Kras(LSL-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 or extracellular signal-regulated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK (extracellular signal-regulated kinase) signaling drives neoplasia in Mx1-Cre, Kras(LSL-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 Kras(G12D) hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasms and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.

Aberrant signal transduction plays a central role in the pathogenesis of myelodysplastic syndrome/ myeloproliferative neoplasm (MDS/MPN), as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the key role of Ras signaling in hematopoietic neoplasms, there are currently no signal transduction inhibitors with established efficacy. This necessitates a screen of inhibitors that may potentially reveal novel therapeutic strategies and inform efforts to treat neoplasms driven by hyperactive Ras signaling. Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 (n= 10) and wildtype (WT) mice (n=18) were utilized in the screening of 94 different inhibitors. A disease relevant, homogenous population of PreGM cells identified as Lineage lo/- Sca1- c-kit+ CD34+ FcγRII/III- CD105- CD150- was purified from bone marrow using flow cytometry. The PreGM cells were then sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1E-7× (5E-7ug/ml) to 1× (5ug/ml). DMSO and cytotoxic agents (Cytarabine, Adriamycin, Gemcitabine) served as negative and positive controls respectively. The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc’ package from the R Project for Statistical Computing. The 94 drug candidates tested in this screen included cytotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. However, none of the compounds tested were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells when compared to WT cells. Furthermore, the IC50s calculated for mutant and WT cells were not significantly different across all inhibitors tested. The majority of compounds tested were either FDA approved drugs or agents used in recent clinical trials. Hyperactive Kras signaling is a major underlying etiology for MDS/MPN, and thus we designed our study to test the efficacy of these candidates against mutant Kras cells. Our results indicate that mutant cells have similar drug sensitivities as normal cells over a broad range of mechanistic approaches. This leads us to believe that "synthetic" lethal opportunities may not be the approach of choice for treating Kras driven neoplasms. However, these findings have directed our efforts towards compounds that target upstream mediators of Kras signaling and future endeavors are currently underway to use the assay system to assess the activity of a highly curated library of kinase inhibitors on mutant and WT proliferation. Citation Format: Wan Xing Hong, Jon Akutagawa, Steven Chen, Michelle Arkin, Benjamin S. Braun. Library screen to rapidly determine activity against normal and mutant bone marrow progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2013-5524

Abstract 4916 Background: Aberrant signal transduction plays a central role in the pathogenesis of MDS/MPN, as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the central role of Ras signaling in the pathogenesis of JMML, at this time there are no signal transduction inhibitors with established efficacy in JMML. A screen of inhibitors has the potential to reveal potential therapeutic strategies and inform efforts to treat other neoplasms driven by hyperactive Ras signaling, both in the hematopoietic system and elsewhere. Aim: To investigate novel therapeutic options for JMML by utilizing a novel, reproducible system for rapid screening in primary cells. Innovations include using flow cytometry to isolate a highly clonogenic, disease-relevant “PreGM” population of primary bone marrow cells that recapitulate the abnormal growth pattern characteristic of JMML and unsorted bone marrow, the use of a genetically engineered mouse model, and the development of automated microscopy protocols. Method: Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 and wildtype mice were utilized in the screens. PreGM cells, identified as Lineage lo/- Sca1- c-kit+ CD34+ CD16/32- CD105- CD150-, were purified from harvested bone marrow using flow cytometry. The purified PreGM cells were sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1X (5 μg/ml, approx. 10 μM for most compounds) to 10−7X (5×10−7 μg/ml). The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions (at 37°C, 98% humidity and 5% CO2) in 80% IMDM, 20% FBS and saturating dose of 10ng/ml of GM-CSF. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 (GE). A total of 94 different inhibitors were screened using this method. The screen included a negative control (DMSO) and cytotoxic positive controls (Cytarabine, Adriamycin and Gemcitabine). Compound families included cyotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. The majority of compounds were either FDA approved drugs or agents used in recent clinical trials. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. Results: Primary bone marrow cells were harvested from a total of 28 mice, 18 wild type (WT) and 10 Mx1-Cre, KrasD12. PreGM growth was quantified and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc’ package from the R Project for Statistical Computing. Out of 94 candidates tested in this screen, none were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells. Neither were any of the drugs found to be comparatively toxic to WT cells or to have significantly higher IC50s in mutant PreGM cells in comparison to WT cells. Some compounds of interest included Vorinostat, an epigenetic inhibitor, which was found to have robust inhibitory activity against both mutant and WT cells. It has comparable IC50s in mutant and WT cells with a calculated IC50 of 0. 0480X (std. error: 0. 135) in Mx1-Cre, KrasD12 cells and 0. 0244X (std. error: 0. 0293) in WT cells. Conclusion: None of the 94 compounds used in the screen were found to preferentially inhibit mutant or WT cell growth, indicating that Kras mutant cells have similar drug sensitivities to normal cells over a broad range of mechanistic approaches. These findings suggest that it may be difficult to find “synthetic lethal” opportunities for drugs that are selectively toxic to primary cells driven by hyperactive Ras signaling. Disclosures: No relevant conflicts of interest to declare.