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The reason why different NRAS mutants are enriched in each tumor type is unclear. We made a suite of conditional, Nras knock-in models (LSL-Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test whether melanocyte transformation is dependent upon functions specific to the NRAS mutants found in human melanoma (Q61R and K). Spontaneous melanomas were rare in mice expressing Nras mutants infrequently observed in human melanoma (G12D, G13D, G13R, Q61H, or Q61P). However, melanocyte-specific Nras Q61R or Q61K expression induced rapid melanoma onset with high penetrance. Cohorts of mice carrying one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P, or -Q allele were used to evaluate potential interactions between Nras mutants. These combinations initiated tumors with kinetics intermediate to those of mice homozygous for either allele (Nras Q61R or 61X) except in the case of Nras Q61Q/Q61R mice, which rarely developed tumors. Genomic and in vitro analyses revealed that the melanoma-initiating potential of each NRAS mutant directly correlated with enhanced MAPK signaling. Together, these data establish that functions specific to the most common NRAS mutants in human melanoma are needed to initiate the disease. Citation Format: Christin E. Burd. Allelic specificity: The key to NRAS-mutant melanoma initiation? [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA09.

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.

Summary There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

There is a growing need to understand how aging influences cancer outcomes in older adults. Prior work shows that older adults with cancer would elect to forgo life-prolonging therapies that would lead to cognitive or physical disability. Here, we hypothesized that aging biomarkers would define individuals at risk for impairment and identify therapeutic regimens associated with accelerated aging. We first tested whether published epigenetic clocks would maintain validity in isolated CD3+ T cells. Potential advantages of using this approach in cancer patients include reduced sample complexity, the ability to survey aging in the context of clonal B cell expansions, and the critical importance of this cell population in cancer control. The Hannum, Horvath, GrimAge, PhenoAge, and PACE clocks increased with chronologic age in CD3+ T cells from 185 healthy donors. However, accelerated epigenetic age varied in PBMCs and CD3+ T cells isolated from the same blood sample. Next, we conducted a longitudinal analysis of adults ≥60 years of age newly diagnosed with non-small cell lung cancer (n=41) or hematologic malignancy (n=94). Standard clinical tools were used to assess fitness (ECOG performance, IADLs, SPPB), comorbidities, cognition (BOMC), and potential for therapeutic toxicity (CARG chemo-toxicity calculator). Relationships between these metrics and baseline CD3+ T cell epigenetic age were investigated. These analyses revealed a direct correlation between epigenetic age and increasing ECOG performance score. Cancer therapies, including high-dose chemotherapy, immunotherapies, and hypomethylating agents, significantly impacted patient epigenetic age. No relationship between outcome and baseline epigenetic age was observed in either population. These data support the use of the Hannum, Horvath, GrimAge, PhenoAge, and PACE clocks to measure aging in CD3+ T cells, though differences in PBMC and CD3+ T cell measurements suggest that the rate of epigenetic aging may differ in each sample type. The inability of epigenetic clocks to consistently measure physical function and cognition or predict patient outcomes implies that new clocks may be required to circumvent the heterogeneity of disease, therapies, and comorbidities in older adults with cancer. Citation Format: Christin E. Burd. The tick-tock of epigenetic clocks in patients with cancer [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA015.

This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR004) of the Conference Proceedings. Citation Format: Daniel Spakowicz, Rebecca Hoyd, Caroline E. Wheeler, Nyelia Williams, Amna Bibi, Marium Husain, Srichandhana Rajamouli, Shankar Suman, Joseph Amann, Madison Grogan, Pooja Vibhakar, Dwight H. Owen, David P. Carbone, Ashley Rosko, Christin E. Burd, Carolyn J. Presley. Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A021.

The gut microbiome changes with age and affects many aspects of human health, including response to cancer treatments. Cancer survival rates have improved with new treatment options, including immune checkpoint blockade (ICB); however, the objective response rate remains low. Manipulation of the microbiome is a promising approach to improving cancer outcomes, but the effect of age is understudied. Here, we sought to understand whether (1) specific microbes are associated with treatment response in older adults with non-small cell lung cancer (NSCLC) and (2) whether these microbes are the same as for younger adults. Next, we explored the causal effects of the microbiome on ICB response in mouse models and the relationship with blood-based markers of T-cell senescence. We conducted a prospective cohort study of adults ≥60 years with a new diagnosis of NSCLC who received any treatment modality. Stool was collected, and metagenomic whole-genome shotgun sequencing was performed. Blood T-cells were isolated, the RNA purified and then assessed for markers of senescence by nanostring. Response to treatment was determined by RECIST v1.1 criteria. Generalized linear regression was used to relate baseline microbiome abundances to treatment response and non-parametric correlations associated with CDKN2A (p16) expression to microbe abundances. To assess the causal role of the gut microbiome in ICB response, we gavaged gut microbiome samples from responders and non-responders into C57BL/6 mice to create human-microbiome avatar models. The mice were then injected with MC38 cancer cells and treated with anti-PD1 or isotype control antibodies, and tumor volume was measured over time. Biospecimens and best response data at three months were captured from 23 patients, of which five had a complete response, eight had a partial response, eight had stable disease, and two had progressive disease. Over 50 microbes were associated with a response after p-value adjustment. Responder stool was enriched for microbes associated with youth and ICB response (Bifidobacterium adolescentis, p = 2.64e-20). However, microbial taxa associated with response differed from those reported in younger populations (Firmicutes sp. CAG 145, p = 1.58e-20, Oscillibacter sp. 57-20, p = 7.96e-24). Stool from non-responders (NRs) was enriched in taxa previously linked to treatment-related toxicities and shorter progression-free survival (Streptococcus lutetiensis, p = 4.55E-24) but also contained microbes previously linked to response in younger adults (e.g., Roseburia sp. CAG 309, p = 5.16e-15). The T cell senescence marker, p16, correlated with the most enriched taxon in non-responders NRs (Streptococcus thermophilus, r = 0.45, p = 0.02), suggesting a connection between immune aging and the microbiome. Preliminary fecal transplant studies in mice showed improved ICB response in mice engrafted with stool from responders versus non-responders. Together, these data identify potential differences in the gut microbiomes of young and older adult NSCLC patients who respond to ICB. Citation Format: Daniel Spakowicz, Rebecca Hoyd, Caroline E. Wheeler, Nyelia Williams, Amna Bibi, Marium Husain, Srichandhana Rajamouli, Shankar Suman, Joseph Amann, Madison Grogan, Pooja Vibhakar, Dwight H. Owen, David P. Carbone, Ashley Rosko, Christin E. Burd, Carolyn J. Presley. Older adult-specific microbes correlate with treatment response and markers of T-cell senescence in NSCLC [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR004.

Background The impact of biologic aging on immune checkpoint inhibitor (ICI) toxicity and efficacy is underexplored in metastatic melanoma (MM). In peripheral blood T lymphocytes (PBTLs), biologic aging is characterized by changes in T‐cell composition and cellular senescence. Whether indicators of PBTL biologic aging vary in MM patients or can be used to predict premature ICI discontinuation (pID) is unknown. Methods We prospectively collected PBTLs from 117 cancer‐free controls and 46 MM patients scheduled to begin pembrolizumab or nivolumab monotherapy. Seventy‐four mRNAs indicative of T‐cell subset, activation, costimulation/inhibition, and cellular senescence were measured by Nanostring. Relationships between each mRNA and chronologic age were assessed in patients and controls. Candidate biomarkers were identified by calculating the hazard ratio (HR) for pID in patients divided into low and high groups based on log‐transformed mRNA levels or the magnitude by which each mRNA measurement deviated from the control trend (Δage). Area under the curve (AUC) analyses explored the ability of each biomarker to discriminate between patients with and without pID at 6 months and 1 year. Results Fifteen mRNAs correlated with chronologic age in controls, including markers of T‐cell subset, differentiation, cytokine production, and costimulation/inhibition. None of these mRNAs remained correlated with age in patients. Median follow‐up was 94.8 (1.6‐195.7) weeks and 35 of 46 patients discontinued therapy (23 progression, seven toxicity, and five comorbidity/patient preference). Elevated pretherapy CD8A (HR = 2.2 [1.1‐4.9]), CD45RB (HR = 2.9 [1.4‐5.8]), and TNFRSF14 (HR = 2.2 [1.1‐4.5]) levels predicted pID independent of Δage‐correction. CD3ε, CD27, and FOXO1 predicted pID only after Δage‐correction (HR = 2.5 [1.3‐5.1]; 3.7 [1.8‐7.8]; 2.1 [1.1‐4.3]). AUC analysis identified Δage‐CD3ε and Δage‐CD27 as candidate predictors of pID (AUC = .73 and .75). Conclusions Correlations between transcriptional markers of PBTL composition and chronologic age are disrupted in MM. Correcting for normal, age‐related trends in biomarker expression unveils new biomarker candidates predictive of ICI outcomes.

Background: Older adults with hematologic malignancy (HM) are a growing demographic and providing effective treatments that balance toxicity and health related quality-of-life (HRQL) is imperative. The well-studied and utilized chemotherapy toxicity tool, the Cancer and Aging Research Group (CARG) chemotherapy toxicity score, has not been validated in hematologic malignancies. Methods: The primary objective of this study was to validate the predictive ability of the CARG score for grade 3-5 toxicity in newly diagnosed (ND) patients >60 years with HM. This was a prospective longitudinal study with 4 study visits: baseline (pre-therapy), visit 2 (Day 90), visit 3 (Day 180), and end-of-study (EOS) which occurred at the earliest of the following events: progression, transplant, or 1-year from baseline. The CARG score was evaluated at baseline. HRQL (PROMIS-GHS) and physical function measured by short physical performance battery (SPPB) were assessed longitudinally at all visits. Treatment toxicity using the NCI CTCAE (version 5.0) were captured monthly, and the worst grade of each type of chemo-related adverse event (AE) was recorded and summarized for each patient. Wilcoxon signed-rank test was used to test if variables changed significantly across visits. Fine and Gray model was used to associate comprehensive geriatric metrics and CARG score with the development of grade 3-5 chemotherapy-related toxicity with death as the competing risk, and Cox model was used to analyze overall survival (OS). Results: Ninety-seven patients with hematologic malignancy (myeloid n=34, lymphoma n=35, plasma cell n=28) were enrolled. The median age was 70 years (range 60-88) with a median 159 days on study (range 1-435). Baseline evaluations: ECOG PS was 0-1 in 69 (85%), median IADL score was 13 (range 5-14), median MOS physical health score was 44.4 (range 0-100), median self-reported KPS was 80% (50-100%), and median comorbidity score was 6 (range 2-12). PROMIS median scores improved from baseline (32, range: 12-49) to EOS (35, range: 16-47, p=0.05). Median SPPB scores improved significantly from baseline (5, range 0-12) to EOS (9, range 0-12, p=0.005). During the study period, 75 patients had 334 grade 1-2 AEs, and 42 patients had 82 grade 3-5 AEs. Hematologic toxicities were more common with 36 (37%) patients having anemia (30 grade 1-2, and 6 grade 3-5) and 11 (11%) patients having febrile neutropenia (all grade 3-5). In multivariable analysis, significant risk factors associated with grade 3-5 toxicity (p Conclusions: The CARG chemotoxicity score was not predictive of grade 3-5 toxicities in patients ND with HM, but was univariably associated with OS. Higher SPPB scores were strongly associated with OS. Future studies, evaluating modifications of the CARG score are indicated for patients with HM. Objective measures of function, such as the SPPB, may be a reliable method to stratify treatment intensities for older adults with HM. Disclosures Mims: Glycomemetics: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Aptevo: Research Funding; Leukemia and Lymphoma Society's Beat AML clinical study: Consultancy, Research Funding; Xencor: Research Funding; Kartos Pharmaceuticals: Research Funding; Genentech: Consultancy; Abbvie: Consultancy; BMS: Consultancy; Kura Oncology: Consultancy; Syndax Pharmaceuticals: Consultancy; BMS: Consultancy; Jazz Pharmaceuticals: Consultancy; Aptevo: Research Funding. Woyach: AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; Gilead Sciences Inc: Other: Data & Safety; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee.

Background: Gauging fitness remains a challenge among older adults with hematologic malignancies, and interventions to restore function are lacking. We pilot a structured exercise intervention and novel biologic correlates of aging using epigenetic clocks and markers of immunosenescence to evaluate changes in function and clinical outcomes. Methods: Older adults (n=30) with hematologic malignancy actively receiving treatment were screened and enrolled in a 6-month exercise intervention, the Otago Exercise Programme (OEP). The impact of the OEP on geriatric assessment metrics and health-related quality of life were captured. Clinical outcomes of overall survival and hospital utilization (inpatient length of stay and emergency department use) in relationship to geriatric deficits were analyzed. Results: Older adults (median age, 75.5 years [range, 62–83 years]) actively receiving treatment were enrolled in the OEP. Instrumental activities of daily living and physical health scores (PHS) increased significantly with the OEP intervention (median PHS: visit 1, 55 [range, 0–100]; visit 2, 70 [range, 30–100]; PP=.05). Quality of life (Patient-Reported Outcome Measurement Information System [PROMIS]) improved significantly by the end of the 6-month period (median [range]: visit 1, 32.4 [19.9–47.7]; visit 3, 36.2 [19.9–47.7]; P=.01]. Enhanced measures of gait speed and balance, using the Short Physical Performance Battery scores, were associated with a 20% decrease in risk of death (hazard ratio, 0.80; 95% CI, 0.65–0.97; P=.03) and a shorter hospital length of stay (decrease of 1.29 days; 95% CI, −2.46 to −0.13; P=.03). Peripheral blood immunosenescent markers were analyzed in relationship to clinical frailty and reports of mPhenoAge epigenetic analysis are preliminarily reported. Chronologic age had no relationship to overall survival, length of stay, or emergency department utilization. Conclusions: The OEP was effective in improving quality of life, and geriatric tools predicted survival and hospital utilization among older adults with hematologic malignancies.

The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.

The Kirsten rat sarcoma (KRAS) proto-oncogene regulates signaling pathways that control proliferation (e.g. MAPK pathway), metabolism and survival (e.g. AKT pathway), and apoptosis (e.g. Hippo pathway). Mutations in KRAS are found in 40% of colorectal cancers (CRC). However, there are no FDA-approved drugs for CRC that target this oncogene. One reason why developing drugs against KRAS is difficult is because a number of different oncogenic alleles have been identified. Thus, an allele-specific approach to target KRAS is necessary. In turn, this approach requires a detailed understanding of their structure and function to drive cell transformation and tissue dysregulation. Our goal is to characterize the function of KRAS A59T in CRC. These alleles are interesting for a number of reasons. First, A59T discriminates retroviral homologues of mammalian RAS from their cellular counterparts. Second, unlike other oncogenic alleles, the KRAS A59T protein undergoes autophosphorylation, the biological function of which is unknown. Third, patient data show that A59 alleles of KRAS frequently co-occur with other genetic alterations in the MAPK signaling pathway, which is uncommon for other KRAS alleles. We hypothesize that KRAS autophosphorylation promotes CRC in a manner that is independent, but cooperative, with the MAPK signaling pathway. To test this hypothesis, we first characterized the function of KRAS A59T and its phosphomimetic A59E using in silico and biochemical techniques, along with classic assays of cell transformation. Next, we generated a new genetically engineered mouse model, K-Ras+/LSL-A59E, in order to test how KRAS autophosphorylation cooperates with other oncogenic mechanisms of MAPK pathway activation to alter colon homeostasis and model colon tumorigenesis. Consistent with our hypothesis, KRAS autophosphorylation and A59E inhibit KRAS-effector interactions that activate the MAPK, AKT, and Hippo signaling pathways. How this change in function alters the ability of K-RasA59E, versus K-RasG12D, to promote hyperproliferation and tumor development in combination with N-RasQ61K and Apc loss in the mouse colon, is currently underway. Ultimately, these studies bring us closer to discovering allele-specific vulnerabilities of KRAS and provide a means to test less understood aspects of oncogenic KRAS signaling. Citation Format: Christian W. Johnson, Elizabeth M. Terrell, Christin E. Burd, Deborah Morrison, Kevin M. Haigis. Characterization of oncogenic KRAS A59 alleles and their cooperation with the MAPK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 84.

Summary To mitigate melanoma risk, sunscreen use is widely advocated; yet, the ability of sunscreens to prevent melanoma remains controversial. Here, we test the tenet that sunscreens limit melanoma risk by blocking ultraviolet radiation (UV)-induced DNA damage using murine models that recapitulate the genetics and spontaneous evolution of human melanoma. We find that a single, non-erythematous dose of UV dramatically accelerates melanoma onset and increases tumor multiplicity in mice carrying an endogenous, melanocyte-specific NRas61R allele. By contrast, transient UV exposure does not alter tumor onset in mice lacking p16INK4a or harboring an NRas12D allele. To block the rapid onset of melanoma cooperatively caused by UV and NRas61R, we employed a variety of aerosol sunscreens. While all sunscreens delayed melanoma formation and blocked UV-induced DNA damage, differences in aerosol output (i.e., amount applied/cm2) caused variability in the cancer preventative efficacy of products with identical sunburn protection factor (SPF) ratings.

Senescent cells within the tumor microenvironment (TME) adopt a proinflammatory, senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression, and therapeutic resistance. Here, exposure to palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA damage–independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure of nontransformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined melanoma cells (i.e., Ras mutant, Braf mutant, and Ras/Braf wild-type). When cultured in the presence of CDK4/6i-induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative responses. However, in vivo, PD-0332991–treated fibroblasts enhanced the growth of all melanoma lines tested and promoted the recruitment of Gr-1–positive immune cells. These data indicate that prolonged CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such senescent cells suppress the antitumor immune response and promote melanoma growth in immunocompetent, in vivo models.Implications: The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences that should be considered in clinical trials aiming to treat melanoma and other cancer types. Mol Cancer Res; 15(3); 237–49. ©2016 AACR.

Aging in mammals is the gradual decline of an organism's physical, mental, and physiological capacity. Aging leads to increased risk for disease and eventually to death. Here, we show that Brd2 haploinsufficiency (Brd2+/-) extends lifespan and increases healthspan in C57B6/J mice. In Brd2+/- mice, longevity is increased by 23% (p

Multiple myeloma (MM) is a disease of aging adults and autologous stem cell transplant (ASCT) is considered the standard of care. As the population ages a growing number of older adults will undergo ASCT and an objective approach to estimate physiologic reserve and transplant morbidity risk is warranted. Here, we evaluate assess p16INK4a (p16), a molecular aging biomarker, along with geriatric metrics to determine risk of transplant toxicity. Methods We prospectively evaluated 100 MM patients for frailty before and after ASCT using a Geriatric Assessment (GA) and collected T-cells for analysis of p16 using a custom nanostring codeset. Results Pre-transplant physical function was predicative of hospital length of stay (LOS). Each one-unit increase in physical function score, the average LOS decreased by 0.52 days (95% CI, −1.03–0.02); p = .04). Similarly, higher self-report of ADL/IADL (Human Activity Profile was associated with shorter LOS (0.65 less days (95% CI −1.15 to −0.15), p = .01). Patients with anxiety/depression (OR = 1.10 (95% CI 1.00–1.22), p = .056), lower handgrip strength (OR = 0.90 (95% CI 0.82–0.98), p = .02), falls (OR = 1.60 (95% CI 1.07–2.38), p = .02), or weight loss (OR = 5.65 (95% CI 1.17–25.24), p = .03) were more likely to be re-admitted. The estimated EFS at 1-year was 85% (95% CI, 75–91) with median follow-up of 15.7 months. Weight loss was a significant predictor of EFS (HR = 3.13 (95% CI 1.15–8.50), p = .03). Frailty assessment by self-reported fatigue minimally correlated with T-cell p16 expression (r = 0.28; p = .02). Age, Karnofsky Performance Status (KPS), or Hematopoietic cell transplantation-specific Co-Morbidity Index (HCT-CI) did not predict hospital LOS or readmissions. Conclusions Our data illustrate that a GA can identify individuals with MM who are at greater risk for morbidity following ASCT.

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional “knock-in” mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation. Cancer Discov; 4(12); 1418–29. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1355

Background: Multiple Myeloma (MM) is associated with T-cell dysfunction. MM-related T-cell deficiencies are reported as both quantitative and qualitative defects with variable functional abnormalities. The clinical significance of these T-cell abnormalities in MM is not fully characterized. Furthermore, the impact of Autologous Stem Cell Transplant (ASCT) on T-cell subsets and immune checkpoint molecules, including PD-L1, PD-1, BLIMP-1, CTLA-4/CD28, TIM-3, and LAG-3, is being explored. Here we examined these features along with other mRNA markers of cellular senescence, immunosenescence, and exhaustion in MM patients pre- and post-ASCT. These studies aimed to improve understanding of the immunologic changes in MM and relationship to disease progression. Evaluating T-cell immune reconstitution post-transplant is invaluable for future therapies to identify targets or stimulate T-cell response to mitigate relapse. Methods: 100 MM patients were prospectively enrolled in a longitudinal study prior to ASCT for analysis of clinical and biologic factors related to clinical outcomes and event free survival (EFS) post-transplant. Paired peripheral blood T-cells (PBTL) were analyzed (n=31) before ASCT and 90 days post-ASCT. PBTL mRNA targets were anayzed using a custom Nanostring platform (OSU_Senescence) evaluating markers of cellular senescence, immunosenescence, and exhaustion. T-cell populations and subsets were further characterized by flow cytometry pre- and post-ASCT in 20 representative trial patients and 10 age- and sex-matched controls. Serum samples were analyzed for soluble ligands using Luminix MAGPIX multiplex analysis. Results: Increased PBTL LAG-3 mRNA expression at 90 days post-ASCT was significantly associated with EFS, HR 5.44 (95%CI 1.92-15.46, p=0.001, adjusted p* controlling for false discovery rate=0.038). When adjusted for age, a similar relationship of increased PBTL LAG-3 mRNA expression and EFS was found, HR 5.66 (95%CI 1.83-17.47, p=0.003, p*=0.056). No relationship was observed between other mRNA markers of immunosenesence or exhaustion and EFS. Flow cytometric analysis of post-transplant PBTLs revealed an inverted CD4:CD8 ratio, reduced CD28 expression on CD8+ T-cells, and increased levels of CD4+ T-regulatory cells. LAG-3 expression was significantly increased in CD4+ T-cells post-ASCT, predominately in the CD4 naïve and central memory subsets. Soluble LAG-3 (sLAG3) serum concentrations were similar pre- and post-ASCT, and in comparison to controls. Conclusions: We found increased expression of Lymphocyte-Activation Gene, LAG-3 (CD223), on CD4+T-cells post-ASCT. PBTL LAG-3 mRNA expression 90 days post-ASCT was associated with EFS and may serve as an early indicator of adverse outcomes. LAG-3 is expressed on activated T-cells and modulates T-cell expansion and function. Future studies targeting the LAG-3 pathway are warranted to restore T-cell dysfunction and augment immunity in MM. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is an incurable hematologic malignancy diagnosed primarily in older adults. As the population ages, myeloma incidence is expected to increase at a higher rate than many other malignancies.1 Approved chemotherapy and targeted regimens for older adults with MM are numerous and exhibit distinct toxicities. The physiological heterogeneity of older adults makes it challenging for physicians to identify the most effective, yet best tolerated regimen, for each MM patient. As such, use of a two-drug regimen, three-drug regimen, or intensive autologous hematopoietic stem cell transplant (AHSCT) is often subjective. Consequently, AHSCT eligibility is subjectively applied and more objective measures are warranted to better understand health status in older adults. We believe that objective markers of physiological age will improve treatment stratification and will be an additional tool in understanding how treatment and transplant have an impact on health status.

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth in vivo. We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. In vitro, selinexor increased PDCD1 and CTLA4 gene expression in leukocytes and induced CD274 gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (P < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (P ≤ 0.050) and CD4+ T cells with a Th1 phenotype (P ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. Mol Cancer Ther; 16(3); 417–27. ©2017 AACR. See related article by Tyler et al., p. 428.

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

e21578Background: Senescence is an irreversible cell cycle arrest associated with sustained p16INK4a (p16) expression and biological aging. The inability of senescent cells to proliferate suggests ...

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.

p16INK4a, located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes. Although it is classically known for its capacity to inhibit cyclin-dependent kinase (CDK) activity, p16INK4a is not just a one-trick pony. Long-term p16INK4a expression pushes cells to enter senescence, an irreversible cell-cycle arrest that precludes the growth of would-be cancer cells but also contributes to cellular aging. Importantly, loss of p16INK4a is one of the most frequent events in human tumors and allows precancerous lesions to bypass senescence. Therefore, precise regulation of p16INK4a is essential to tissue homeostasis, maintaining a coordinated balance between tumor suppression and aging. This review outlines the molecular pathways critical for proper p16INK4a regulation and emphasizes the indispensable functions of p16INK4a in cancer, aging, and human physiology that make this gene special. Mol Cancer Res; 12(2); 167–83. ©2013 AACR.

Of the four molecular subtypes of melanoma (BRAF, NRAS, NF1, and triple WT), NRAS mutant tumors are notoriously more aggressive and challenging to treat. The frequent occurrence of NRAS mutations in benign, congenital nevi suggests that these genetic alterations are early events in melanoma genesis. Since NRAS mutant nevi can remain indolent for years, or even a lifetime, secondary genetic events must be required to drive melanoma initiation. Ultraviolet (UV) light, a major risk factor for melanoma, is known to directly damage DNA and alter the skin microenvironment. Therefore, we hypothesized that the cell-extrinsic and intrinsic events triggered by UV exposure would cooperate with NrasQ61R mutations to drive melanoma formation in vivo. To test this hypothesis we employed a genetically engineered mouse model (i.e. TpN61R) that combines CRE-inducible expression of NrasQ61R with the loss of p16INK4a in melanocytes. Following neonatal CRE induction, we exposed TpN61R mice to a single, nonerythemic dose of UVB radiation and monitored them for the development of melanoma. Exposure to UVB radiation dramatically reduced the melanoma-free survival of TpN61R mice by 80% and increased tumor multiplicity (average 1.2 to 3.4 tumors/mouse); however, it had no impact on tumor growth rates, overall skin proliferation, immune infiltration, or vascularity. To test the respective roles of NrasQ61R and p16INK4a loss in UV-induced melanoma genesis, we generated mice with either melanocytic NrasQ61R expression or p16INK4a loss. Mice with p16INK4a loss alone did not develop tumors in the presence or absence of UV. By contrast, in UV treated TN61R mice, NrasQ61R was sufficient to initiate melanoma formation, albeit with a 66% longer latency than UV exposed TpN61R animals (9.14wks vs. 5.5wks). To determine if NrasQ61R mutations had to be present at the time of UV exposure to drive early melanoma formation, TpN61R mice were exposed to UV prior to CRE activation. Even when NrasQ61R expression was induced three days after UV exposure, melanoma formation was rapidly accelerated. Therefore, NRAS mutations do not need to be present at time of UV exposure to promote early melanoma formation. Genomic analyses comparing spontaneous and UV-induced TpN61R melanomas failed to uncover common secondary mutations that explain the exquisite cooperativity between NrasQ61R and UV light in driving melanoma formation. For this reason, we sought to identify UV-induced, cell-extrinsic factors that might facilitate the initiation of NrasQ61R mutant melanomas via cytokine profiling. Through these analyses we identified UV-induced pro-inflammatory cytokines that could be therapeutically targeted to limit the initiation of NRAS-mutant melanomas. Together, this work explains why UV exposure is such a significant risk factor for melanoma and provides original mechanistic insight into how this deadly disease might be prevented. Citation Format: Rebecca C. Hennessey, Andrea M. Holderbaum, James E. Gillahan, Conor Delaney, Xing Tang, Raleigh Kladney, Christin E. Burd. Ultraviolet light cooperates with NrasQ61R mutations to drive malignant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2685.

Cellular senescence, a process in which cells permanently exit the cell cycle, has pleiotropic biological effects. Several lines of evidence suggest that senescent cells within the tumor microenvironment can elicit a pro-inflammatory secretome that promotes cancer initiation and progression. This proinflammatory phenotype differs amongst senescent cells triggered by distinct stimuli; therefore, multiple forms of senescent cells likely exist within the tumor stroma during cancer evolution and treatment. Here, we sought to determine how stromal senescence initiated by clinically relevant treatments would influence melanoma growth. To generate senescent stromal populations, we treated presenescent MEFs (p4) with ultraviolet light (UV), Mitomycin C (MMC), or a CDK4/6 inhibitor (CDK4/6i). In contrast to p4 fibroblasts, cells treated with UV, MMC or CDK4/6i exhibited cell cycle arrest, increased SA-β-gal positivity and elevated p16INK4a expression. Pronounced 53BP1 and γH2AX foci were only observed in the UV and MMC treated cells, suggesting the CDK4/6i treatment did not induce DNA damage. Profiling of gene expression using a TaqMan mouse inflammation expression panel revealed distinctions amongst the 4 fibroblast lines. Of the senescent cell populations, CDK4/6i fibroblasts showed the highest number of elevated pro-inflammatory transcripts. To define the in vitro paracrine growth effects induced by stromal senescence, melanoma cell lines harboring either Braf600E, Ras, or unknown (wild-type) driver were co-cultured with each fibroblast population. In general, melanoma cells grew similarly on senescent and non-senescent fibroblasts; however, tumor cell growth was genotype-dependent. Notably, CDK4/6i fibroblasts never stimulated additional tumor cell growth in vitro. In contrast, syngeneic transplantations of fibroblasts and tumor cells into immune competent mice revealed that all senescent fibroblasts stimulated tumor growth in vivo. However, whether senescent and non-senescent fibroblasts promoted tumor growth at the same rate was genotype-dependent. Since this is the first study to examine the paracrine effects of senescent stromal cells using an immune competent mouse, we performed IHC for CD45 to assess whether immune cell recruitment differs in the presence of senescent stromal fibroblasts. No increase in CD45 positive infiltrates was observed in the melanoma lines co-injected with CDK4/6i fibroblasts. Together, this work reveals that melanoma growth in response to senescent stromal fibroblasts is genotype dependent, and that this stromal promotion of cancer growth can be accurately assessed only in the context of an immune competent, syngeneic host. Moreover, these data suggest that stromal senescence induced by emerging CDK4/6 inhibitor therapies and/or regimens combining chemotherapy/radiation with immune checkpoint blockade may also promote tumor growth through the paracrine effects of senescent bystanders. Citation Format: Xiangnan Guan, Kyle Lapak, Rebecca Hennessey, Christin Burd. PD0332991-induced stromal senescence promotes melanoma growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2827.

Cutaneous melanoma claims the life of one American every hour, and while the etiology of this tumor type is not entirely understood, exposure to ultraviolet (UV) radiation (280-340nm) is a major risk factor. For this reason, the use of UV-blocking sunscreens is strongly advocated; however, few studies have tested the relative efficacy of these agents in preventing melanoma formation in vivo. Here, we employed a new genetically engineered mouse model (TpN61R) to examine the ability of 6 chemically distinct SPF30 sunscreens to prevent melanoma. In this TpN61R model, topical 4-hydroxytamoxifen (4OHT) treatment induces the melanocyte-specific expression of oncogenic NRas as well as inactivation of the p16INK4a tumor suppressor. These genetic lesions co-occur in ∼24% of all human melanomas, making the TpN61R model biologically relevant. Since NRAS mutations are an early and UV-independent event in human melanoma, TpN61R mice were first painted with 4OHT and then exposed to a single dose of 4.5 kJ/m2 UVB light one day later. Exposed skin from TpN61R mice showed transient cyclobutane pyrimidine dimer (CPD) formation, however no evidence of edema or inflammation was observed. Despite these mild effects, UVB exposure reduced the melanoma-free survival of TpN61R mice by 80% and increased tumor incidence rate from 1.2 to 3.4 tumors/mouse. Further experiments using a variety of UVB doses (0.25, 1.0, 2.3 and 9.0 kJ/m2) in the TpN61R model revealed a dose-dependent increase in early melanoma incidence. Together, these data establish the exquisite cooperation of UV light and oncogenic NRas mutations in driving melanoma. Taking advantage of this unique model, we tested the preventative efficacy of SPF30 sunscreens with differing chemical composition. Sunscreens components included UVA (avobenzene), UVB (homosalate, octisalate) and broad spectrum (oxybenzone, octocrylene, zinc oxide) blocking agents. Application of sunscreen prior to UVB exposure decreased DNA damage, delayed melanoma onset and reduced tumor incidence in a sunscreen-dependent manner. Thus, SPF30 sunscreens do not equally prevent UVB-induced, NRAS mutant melanoma. This work establishes the first in vivo system to test sunscreen efficacy in NRAS-driven melanoma and will help direct the development of improved melanoma preventatives. Citation Format: Andrea M. Holderbaum, Rebecca C. Hennessey, James E. Gillahan, Anamaria Bonilla, Conor Delaney, Raleigh D. Kladney, Kathleen L. Tober, Tatiana M. Oberyszyn, Christin E. Burd. In vivo modeling of NRAS-mutant melanoma reveals differential preventative efficacy amongst SPF30 sunscreens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 900.

Melanoma mortality is directly linked to its profoundly metastatic nature and inherent resistance to conventional chemotherapy. For this reason, increased understanding of the molecular mechanisms driving melanoma progression is of utmost importance. The p21-activated serine/threonine kinases (PAKs) are frequently mutated or overexpressed in cancer and have recently been implicated in tumor metastasis, proliferation, and apoptotic resistance. This observation has inspired drug discovery efforts to limit PAK activity; yet, mechanistic understanding of how PAKs (especially Type II PAKs) contribute to tumor initiation and progression is still evolving. PAK7, a type II PAK, is mutated in 16-18% of all melanomas. Melanoma-associated PAK7 mutations occur throughout the gene and have unknown biochemical and physiological consequences. The objective of this study was to determine how tumor-associated PAK7 variants mechanistically contribute to melanoma initiation and progression. First, we characterized PAK7 protein expression in a panel of 39 melanoma cell lines and primary melanocyte cultures. PAK7 levels were similar in both tumorigenic and non-tumorigenic cells and failed to correlate with the status of common melanoma driver mutations (e.g. NRASQ61, BRAFV600). In an array of 101 human tumor biopsies, PAK7 expression did not significantly change with advancing melanoma stage; however, PAK7 levels were higher in regional lymph node biopsies when compared to primary skin lesions. Since mutation is the predominant mechanism affecting PAK7 in melanoma, we investigated the biochemical and physiological consequences of common PAK7 variants. 11 distinct PAK7 mutations were stably expressed at low levels in VMM39 (melanoma) and NCI-H441 (lung) cells. Using these clones, changes in the phosphorylation of downstream PAK7 targets associated with cellular migration (p120), proliferation (CRAF), and apoptotic resistance (BAD, MDM2) were examined. In addition, we defined phenotypic alterations in cellular morphology, proliferation, migration, and apoptotic resistance associated with the expression of each mutant. These data allowed us to classify PAK7 variants of unknown significance into groups associated with specific cellular outcomes. To our knowledge, this is the first study to functionally characterize a wide variety of melanoma-associated PAK7 mutations and represents a critical first step in understanding how this kinase contributes to tumor initiation and progression. Citation Format: Kyle M. LaPak, Michael A. Gross, Denny C. Vroom, Greg B. Lesinski, William E. Carson. Defining the role of PAK7 variants in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 195.

Immunotherapy and immune-based anti-cancer molecules represent a valid strategy to fight cancer. However, the choice of tumor-specific surface molecules for the selective targeting of cancer cells still represents a critical step in the study design for the development of new therapeutic approaches. Notably, the development of phage-display technology for the selection of fully human single chain antibody fragments (scFvs) and complete antibodies directed toward tumor-associated antigens has represented a significant advancement for immunotherapy. Nucleolin (NCL) is one of the most abundant non-ribosomal proteins in the nucleolus. NCL is frequently up-regulated in cancer and in cancer-associated endothelial cells compared to normal tissues, where it is also present on the cell surface. Altered NCL expression and localization results in oncogenic effects such as stabilization of oncogenic mRNAs and microRNAs (miRNAs). Particularly, we demonstrated that NCL enhances the maturation of specific miRNAs (including miR-21, miR-221 and miR-222) causally involved in cancer pathogenesis, aggressiveness, metastatic potential and resistance to several anti-neoplastic treatments. Because of its oncogenic role and specific expression on cancer cell surface, NCL represents an attractive target for anti-neoplastic therapies. To produce a new anti-NCL molecule with significant potential for clinical applications, we took advantage of phage-display technology to isolate a fully human single chain Fragment variable (scFv), named 4LB5, which binds with high affinity to the RNA binding domain (RBD) of NCL. In our previous study we demonstrated that 4LB5 binds NCL on the surface of aggressive breast cancer cells and inhibits their proliferation both in vitro and in vivo, representing the prototype of a new class of immune-based anti-NCL compounds. Since NCL expression has been previously reported on the cell surface of skin cancer cell lines and up-regulation of NCL-dependent microRNAs was described in human melanomas, the objective of this project was the assessment of 4LB5 as a potential tool for melanoma therapy. To this aim, the recombinant scFv was expressed as His6-fusion protein in E.Coli and purified by affinity chromatography, as previously described. By using Enzyme-Linked Immunosorbent Assays (ELISA), we demonstrated a significant binding of 4LB5 to the cell surface of different melanoma cell lines of both human and mouse origins. Notably, inhibition of NCL expression by siRNA transfection reduced the binding of 4LB5 to the cell surface of these cell lines, further supporting its specificity against NCL. Then, we assessed the potential effects of 4LB5 treatment on cell proliferation. Colony formation assays demonstrated that 4LB5 significantly affected cell proliferation of both human and mouse melanoma cell lines. Our results, in agreement with previously reported data, further support the potential activity of 4LB5 as a tool for cancer therapy, paving the way for additional investigations aimed to fully elucidate the molecular mechanisms affected by this scFv and resulting in its anti-neoplastic therapy in human melanomas. Furthermore, this study supports the idea that anti-NCL immunoagents might represent a class of new anti-cancer compounds with a strong clinical relevance for a wide range of human tumors. Citation Format: Ashley Braddom, Timothy Richmond, Tyler Sheetz, Erika Reese, Anna Tessari, Kathleen Tober, Christin E. Burd, Claudia De Lorenzo, Edward W. Martin, Jr., Vincenzo Coppola, Michael F. Tweedle, Tatiana Oberyszyn, Carlo M. Croce, Dario Palmieri. Human anti-Nucleolin recombinant immunoagents as new potential tools for melanoma treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C171.

Exportin-1 (XPO1) is a nuclear export protein with >220 cargo proteins, including tumor suppressors and cell cycle modulators. Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >900 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. Selinexor blocks nuclear export of NFAT1c, STAT1 and STAT3, which are implicated in regulating the inhibitory T cell receptor PD-1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and that combination treatment with anti-PD-1 or anti-PD-L1 would thereby enhance the ability of selinexor to elicit antitumor activity. Selinexor increased PD-1 gene expression by ~2-fold in normal lymphocytes and induced PD-L1 gene expression in tumor cell lines. Mice bearing syngeneic colon tumors (colon26) treated with selinexor and anti-PD-1 for 2 weeks demonstrated a significant reduction in tumor growth rate (P < 0.05), while monotherapy with either agent had no significant effect on tumor growth. Similar results were obtained in mice bearing syngeneic B16F10 melanoma tumors, whereby combined treatment with selinexor + anti-PD-1 was superior to either single agent alone (p < 0.034). Combined therapy of mice bearing B16F10 tumors with selinexor and anti-PD-L1 was similarly effective, with significantly smaller tumors at the study endpoint (p < 0.001). No weight loss or signs of toxicity were evident in any in vivo study. Immunophenotypic analysis by flow cytometry revealed that selinexor alone or in combination with anti-PD-1/anti-PD-L1 significantly increased the percentage of splenic NK cells (p≤0.050), while selinexor ± anti-PD-L1 significantly increased the percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p < 0.001). These data indicate that the efficacy of selinexor may be enhanced by disrupting the pre-existing PD-1/PD-L1 signaling in effector cells (T and NK cells). Altogether, these data suggest that the efficacy of selinexor in combination with anti-PD-1 or anti PD L1 in mouse syngeneic tumor models may be due to both disrupting immunosuppressive PD-1/PD-L1 signaling and increasing the frequency of potentially tumor reactive NK cells and Th1 T cells. This provides a rational basis for this treatment combination as a novel therapeutic approach for advanced cancer.

Multiple myeloma (MM) is an incurable hematologic malignancy primarily of older adults. MM treatment decisions are based partially upon chronologic age, a measure that may not reflect the patients’ physiologic age or ability to tolerate therapy. Recently, investigators have established methodology to better quantify biologic age using the molecular marker, p16INK4a (p16). p16 expression is a marker of cellular senescence and is strongly associated with gerontogenic models. p16 rises exponentially with chronologic aging and is influenced by external factors such as physical activity, tobacco use, and solid tumor chemotherapy. Additionally, single nucleotide polymorphisms located near the p16-encoding INK4/ARF locus are linked to age-related diseases (e.g. cardiovascular disease, diabetes, glaucoma) and decreased physical function. p16 mRNA can be measured in peripheral blood T-cells thus providing a practical means of quantifying this molecular marker of age. To determine the effects of MM therapy and autologous hematopoietic stem cell transplant (AHSCT) on biologic aging, we serially measured T-cell p16 expression in MM cohorts and aged matched controls. Methods: 23 MM patients were divided into cohorts of newly diagnosed (ND; n=11) and prior treatment (T; n=12). T-cell isolation was performed using techniques described by Liu and colleagues (Liu et al. Aging Cell 2009); p16 expression was measured using quantitative RT-PCR. 16 MM patients underwent sequential testing, with a median 42 days between each measurement. During this time, 8 patients had no intervention and 8 received immune modulatory (IMiD) therapy. Healthy age matched control T-cells were analyzed for comparison (n=17). Results: MM patients with a median age of 62 were evaluated; 12 with early stage ISS disease, 21 with an ECOG performance status of 0-1, and 6 with high risk FISH abnormalities. 12 MM patients had prior treatment, most included AHSCT (n=11). MM treated patients had several lines of therapy; half with 1-2 lines and half with 3+ lines. Median time from AHSCT to p16 analysis was 1003 days (range 49-3630) and transplant date was unrelated to p16 expression (p=0.9577). The healthy control median age was 60 (SD 11.77) with a range of 35-82 years. T-cell yield among MM patients and healthy controls was not significantly different. Using univariate linear regression, age correlates significantly with p16 expression in healthy controls (p=0.0039) wherein p16 is estimated to increase by 1.035 fold each year [0.05 Cts per year]. Using multivariable regression analysis, age and MM status (ND and T), were both independently associated with p16 expression. When controlling for age, patients previously treated for MM had p16 expression that was ~3.68-fold higher than healthy controls (p=0.0003). There was no significant difference in p16 expression between ND MM patients and healthy controls (fold-change=1.15, p=0.70). MM p16 levels were significantly higher in patients with prior therapy (p=0.0439) and for those who had underwent AHSCT (p=0.0129). However, p16 was unrelated to other clinical factors such as cardiovascular disease, tobacco use, radiation, stage, absolute lymphocyte count, or renal function. 8 MM patients where p16 expression was measured pre and post-IMiD treatment, a significant change in expression was not observed (median percent change: 1.16%, p=0.11); in another 8 MM patients who were untreated, p16 levels were also unchanged over time (median percent change: -0.91%, p=0.38). Conclusions: AHSCT significantly impacts p16 expression in T-cells from MM patients. Specifically, MM patients post-AHSCT show a 3.68 fold increase in p16 expression compared to age-matched controls. Time from transplant to assessment does not impact p16 measurements, suggesting that cellular senescence of the T-cell compartment following AHSCT is persistent. MM patients exhibit known T-cell defects including variable T-cell frequency, abnormal signal transduction, and impaired cytokine secretion. Moreover, MM AHSCT patients also show impaired immune reconstitution with changes in T-cell frequency and function. To our knowledge, this is the first report describing cellular senescence post-transplant. Health status and function are affected by AHSCT; our work is important in understanding how biologic markers of age change with intensive therapy, such as AHSCT, to better predict outcomes in future studies. Disclosures Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

Prostatic adenocarcinoma is the most frequently diagnosed malignancy in the United States and is the second leading cause of cancer deaths among men (Landis et al., 1998). Most prostate cancers are treated by androgen ablation, since androgen is a required mitogen for the survival and proliferation of prostate cells (Cunha et al., 1987; Jenster, 1999; Klotz, 2000; Labrie, 2000). This treatment is initially effective, as upon androgen withdrawal most prostate cancer cells undergo cell cycle arrest or cell death (Denmeade et al., 1996; Murphy et al., 1991; Westin et al., 1995). Unfortunately, androgen independent tumors arise and lead to patient morbidity. Determination of how androgen exerts its effect as a mitogen in early prostate cancer, and how the androgen requirement is subverted in advanced disease is the focus of intensive research.

RB, p107, and p130 are highly related proteins, each capable of regulating cellular proliferation. However, only RB is frequently mutated in cancer. In this issue of Cancer Cell , Chicas et al. shed new light on this conundrum, defining a "special," nonredundant role for RB in promoting cellular senescence.

In human cancer, N-RAS mutations resulting in constitutive, oncogenic signaling are predominately localized to codons 12, 13 or 61. Traditionally, activating RAS mutations have been considered oncogenically equivalent, yet recent studies suggest important clinical distinctions between colon cancers containing K-RAS codon 12 vs. codon 13 mutations. More than 20% of human melanomas harbor N-RAS mutations, the vast majority of which target codon 61, as opposed to N-RAS mutations of codon 12 or 13, which are common in other tumor types. To address this issue, we characterized syngeneic knock-in mouse models conditionally expressing either N-RasG12D or N-RasQ61R under the control of the endogenous locus. In primary melanocyte cultures, activation of either N-Ras allele reduced cellular proliferation even in the presence of concomitant p16INK4a deletion. Correspondingly, melanocyte-specific N-RasG12D expression combined with p16INK4a loss failed to efficiently promote melanoma formation in mice. In contrast, melanocyte-specific N-RasQ61R induction readily combined with p16INK4a loss to promote melanoma formation in vivo with short latency and high penetrance. These results provide a first murine model of melanoma featuring knock-in N-Ras mutationQ61R, and suggest that the preference for codon 61 mutations in human tumors is conserved in mice. Citation Format: Christin Burd, William R. Jeck, Kailing Fu, Kelly S. Clark, Jessie C. Xiong, George P. Souroullas, Norman E. Sharpless. Differential oncogenicity of N-RAS mutations in melanoma. [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 1087. doi:10.1158/1538-7445.AM2013-1087

The p16INK4a tumor suppressor is upregulated and serves to limit proliferation in response to cellular stressors and oncogenic events. Induction of p16INK4a is highly dynamic during tumorigenesis and aging, ranging from undetectable in unstressed cells to >105 mRNA copies per cell when maximally activated. This property, along with the well established activation of p16INK4a in response to a variety of oncogenic stressors, led to the hypothesis that p16INK4a could be an excellent marker of tumorigenesis and aging. Toward that end, we developed a novel knock-in mouse model, p16-LUC, wherein the endogenous p16INK4a promoter drives single-copy expression of firefly luciferase. Using homologous recombination in ES cells, the open reading frame of firefly luciferase was inserted in-frame at the transcriptional start site of p16INK4a, preserving the structure and regulation of other genes (p19ARF and p15INK4b) encoded by the Cdkn2a/b locus. Murine embryo fibroblasts (MEFs) derived from p16-LUC animals demonstrate marked (>105) upregulation of luciferase activity with culture, paralleling that observed for the endogenous allele. Such induction can also be monitored dynamically in vivo following treatment with DNA damaging stressors such as ionizing radiation. To test the ability of this allele to serve as a biomarker of oncogenic stress, we crossed the p16-LUC mouse to numerous genetically engineered mouse models (GEMMs) of human cancer. In these mice, potent induction of luciferase activity was apparent in tumors driven by a wide variety of oncogenes. Moreover, weeks before visual or palpable tumors could be detected, p16-LUC expression localized to future tumor sites. Treatment of these mice with a wide range of chemotherapeutic compounds also revealed novel insights into therapeutic efficacy and potential toxicities. Overall, the p16-LUC model has potential to advance the study of de novo tumor formation, therapeutic efficacy and toxicology by providing a reagent to conduct long-term, in vivo studies of GEMMs and potential anti-cancer compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5271. doi:10.1158/1538-7445.AM2011-5271