Citations

Abstract Objective QTc interval prolongation can lead to life-threatening complications such as torsade de pointes (TdP), ventricular tachycardia (VT), and sudden cardiac death (SCD). It can occur with various tyrosine kinase inhibitors (TKIs) but comparative analyses on the incidence and complication rates are scarce. We thus conducted a comprehensive analysis of TKI use and QTc prolongation in clinical practice. Methods We retrospectively reviewed the electronic medical records of all cancer patients who were treated with TKI between 01/2005 and 12/2018 at our institution. QTc prolongation was defined as a QTc ≥450 ms or 460 ms among male or female patients, respectively. For each type of TKIs, we determined the administration rate and incidence of QTc interval prolongation. We also studied the frequency of QTc prolongation ≥500 ms, the rate of increase of the QTc interval by ≥60 ms, and the development of complications (VT, TdP, and SCD). Results In the present study, we analyzed the data of 685 cancer patients (431 male and 254 female), including 299 patients with RCC, 188 with chronic leukemia, 55 with acute leukemia, 65 with thyroid cancer, 48 with lung cancer and 39 with GIST. These patients received 902 TKI administrations and QTc prolongation was reported in 1/3 of these (289 administrations). The highest frequency was seen with imatinib, nilotinib, and dasatinib (30, 40 and 50%). Among cases of QTc prolongation, a QTc interval ≥500 ms was documented in 53 (18.3%) and QTc progression ≥60 ms in 72 (25%). Complications were found in 14 cases (5%) including VT in 9, TdP in 2 and SCD in 3 administrations. Table 1 demonstrates the findings for each TKI. Findings for TKIs in all patients Total Prolonged QTc QTc ≥500 QTc progression ≥60 VT SCD TdP Imatinib 165 54 13 10 2 Nilotinib 75 33 8 19 Dasatinib 115 58 10 16 2 1 Sunitinib 134 31 1 2 1 1 Pazopanib 165 36 5 6 2 1 Others 248 77 16 18 2 1 1 Conclusion The current findings suggest that TKI therapy leads to QTc prolongation in 1/3 of patients on average and most commonly with the Bcr-Abl TKIs, imatinib, nilotinib, and dasatinib. While SCD is rare (1%) it can still evolve and in 5% of all QTc prolongations with TKIs are potentially life-threatening. These data support recommendations for serial ECGs in cancer patients undergoing TKI therapy. Acknowledgement/Funding None

Abstract The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients. PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a "PRMT1 signature". This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance. Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5). In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. Disclosures Khaled: Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

Abstract ROR1 is a type-1 tyrosine kinase-like orphan-receptor that ordinarily is expressed during embryogenesis, but that also is found on leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). In prior studies we found ROR1 served as a receptor for Wnt5a, which could promote survival/growth of CLL cells. We found Wnt5a in the plasma is significantly higher in pts with CLL (3.2±1.6 ng/ml (mean ±S.D.), N = 36) than in healthy adults (0.14±0.16 ng/ml, N=14, p<0.01) and also may be elaborated by accessory cells in the CLL microenvironment. On the other hand, reducing expression of ROR1 on CLL cells via siRNA adversely affected leukemia-cell survival. We hypothesized that expression of ROR1 in CLL could play a role in the pathogenesis and/or progression of this disease. Early studies failed to define differences in the expression level of ROR1 on CLL cells of different pts in a small cohort. However, upon further analyses, we observed heterogeneity in the expression levels of ROR1 among samples of different pts in larger cohort. We investigated whether the level of ROR1 expression was associated with disease progression in a cohort of 1,568 CLL pts followed by CLL Research Corium (CRC). CLL samples were uniformly examined for the expression of ROR1 using the same preparation of an Alexa-647-conjugated anti-ROR1 mAb (4A5) and a standardized procedure by the CRC Tissue Core. We used 10 CLL cases, and tested ROR1 expression at 3 different time-points for each case. We found ROR1 expression was stable over time in all CLL cases. Then, we randomly segregated pt samples into either of two cohorts, one a training set of 797, and the other a validation set of 773 cases. For the 797 cases in the training dataset, we used the profile-likelihood method in a Cox regression model of treatment-free survival (TFS) from diagnosis to determine the optimal threshold level of ROR1 expressionthat could segregate pts into 2 subgroups. In the training cohort, 294 pts were stratified into a "ROR1-Low" subgroup and 503 pts were assigned to a "ROR1-High" subgroup, defining a threshold absolute mean fluorescence intensity of CLL cells stained for ROR1 of 29. The subgroup of pts in the ROR1- High subgroup had a median TFS of 6.0 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.0 years. We applied this threshold to segregate the 773 samples of the validation cohort into ROR1-High and ROR1-Low subgroups. We found that high-level expression of ROR1retained its strong predictive adverse effect on TFS in this validation set. The subgroup of pts in the ROR1- High subgroup had a median TFS of 5.7 years, whereas those pts in the ROR1-Low subgroup had a median TFS of 11.8 years. The immunoglobulin heavy-chain variable region (IGHV) mutation status was known for 593 of the 797 samples in the training set (74%) and 586 of the 773 samples in the validation set (76%). A significantly lower percentage of the ROR1-Low cases (34%, or 69 of 203) used unmutated IGHV than did the ROR1-High cases (54%, or 209 of 390) (p<0.001). Nonetheless, using a multivariate Cox regression model with either a non-delayed entry or a delayed entry, we observed that high-level expression of ROR1 had predictive value for short TFS (non-delayed entry HR 1.4, p<0.05; delayed entry HR 1.9, p<0.001) even when the IGHV mutation status was taken into consideration (non-delayed entry HR 2.8, p<0.001; delayed entry HR 2.7, p<0.001). Taken together, this study demonstrates that there is heterogeneity in the expression of ROR1 on CLL B cells. Furthermore, we find that a high level expression of ROR1 is associated with early disease progression in pts with CLL. Table 1. Number of cases with high versus low ROR1 and mutated versus unmutated IGHV in the training and validation cohort Training Dataset Validation Dataset ROR1 MFI distribution (Median) 0~475 (35.5) 0~424(34.6) ROR1Low with Mutated IGHV 139 132 ROR1Low with Unmutated IGHV 64 84 ROR1High with Mutated IGHV 181 156 ROR1High with Unmutated IGHV 209 214 Total With Known IGHV Mutation Status 593 586 Disclosures Keating: Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Other: Advisory board. Kay:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Byrd:Acerta Pharma BV: Research Funding. Gribben:Celgene: Consultancy, Honoraria; Janssen: Honoraria; Roche/Genentech: Honoraria; Pharmacyclics: Honoraria; Gilead: Honoraria. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Abstract Background Ixazomib, the first oral proteasome inhibitor, is approved by the US FDA, in combination with Rd, for the treatment of MM patients (pts) who have received at least 1 prior therapy. Approval was based on the double-blind, placebo-controlled phase 3 TOURMALINE-MM1 study (NCT01564537) in 722 pts with RRMM (Moreau et al, N Engl J Med 2016), in which IRd showed superior PFS versus placebo-Rd (HR 0.74) at the final prespecified analysis, with limited additional toxicity. Prespecified subgroup analyses showed a consistent PFS benefit with IRd, but with a differential treatment effect according to prior therapy exposure. An exploratory study objective was to evaluate potential relationships between treatment outcomes and tumor gene expression patterns. c-MYC is a proto-oncogene that encodes a transcription factor regulating cell proliferation, growth, protein translation, metabolism, and apoptosis, and increased c-MYC expression is involved in MM pathogenesis/progression. We analyzed the impact of c-MYC expression on PFS in RRMM pts treated with IRd or placebo-Rd. Methods Pts with RRMM after 1-3 prior lines of therapy were randomized 1:1 to receive oral ixazomib or placebo, plus lenalidomide and dexamethasone, until disease progression or unacceptable toxicity. Randomization was stratified by number of prior therapies (1 vs 2-3), proteasome inhibitor exposure, and ISS disease stage. The primary endpoint was PFS by independent review committee assessment. Data reported here are from the final prespecified statistical analysis of PFS (median follow-up ~15 months). Bone marrow aspirate samples were collected at screening for tumor gene expression analysis. Whole transcriptome sequencing (RNAseq) of bone marrow-sorted CD138+ cells was performed using the Illumina Truseq protocol on the Illumina HiSeq platform; this generated a median of 50 million paired-end reads (100 bp length) per sample. c-MYC expression was evaluated by treatment arm and according to number of prior therapies (1 vs 2-3). PFS was analyzed in subgroups dichotomized according to median c-MYC expression (c-MYC-high/c-MYC-low). Results RNAseq data were available for 59% (427/722) of enrolled pts; 213 (50%) had high and 219 (50%) had low c-MYC expression (dichotomized according to the median). Impact of c-MYC expression (high vs low) on PFS, irrespective of treatment arm, was not significant (p=0.98). However, a significant treatment effect of IRd vs placebo-Rd was only seen in pts with high c-MYC expression (interaction p=0.024); in these pts (IRd: n=109; placebo-Rd: n=104) a significant PFS benefit was seen with IRd (HR 0.41; 95% CI: 0.26-0.65, p=0.00011). Impact of c-MYC expression was then analyzed by number of prior therapies. In the overall TOURMALINE-MM1 population, per stratification, 212/213 pts in the IRd/placebo-Rd arms had 1 prior therapy, and 148/149 had 2-3 prior therapies. After median follow-up of ~15 months, median PFS in pts with 1 prior therapy was 20.6 vs 16.6 months with IRd vs placebo-Rd (HR 0.88; 95% CI: 0.65-1.20), and in pts with 2-3 prior therapies was not reached vs 12.9 months (HR 0.58; 95% CI: 0.40-0.84, p=0.0033). The level of c-MYC expression was shown to be significantly higher in pts with 2-3 prior therapies vs 1 prior therapy, with median expression of 6.94 (SD=1.71, n=177) vs 6.52 (SD=1.74, n=250) (log2 scale), t-test p-value 0.0153. In pts with 1 prior therapy, no differences in PFS were observed between pts with low c-MYC expression (HR 1; 95% CI: 0.59-1.8, p=0.934), while a prolonged PFS benefit was observed in pts with high c-MYC expression (HR 0.48; 95% CI: 0.26-0.88, p=0.0146) (Figure, A and B). In pts with 2-3 prior therapies, both the c-MYC-high and c-MYC-low pts showed improved PFS with IRd vs placebo-Rd; the difference in PFS was statistically significant only in c-MYC-high pts (HR 0.36; 95% CI: 0.18-0.72, p=0.00269) (Figure, C and D). Conclusions In TOURMALINE-MM1, c-MYC expression was higher in MM tumor samples from pts with 2-3 vs 1 prior therapies. The IRd regimen was active in c-MYC-high and c-MYC-low pts, but its impact on PFS vs placebo-Rd was greater in c-MYC-high pts, thus providing a potential explanation for the enhanced PFS benefit with IRd vs placebo-Rd in pts with 2-3 prior therapies. The PFS benefit with IRd in c-MYC-low pts with 2-3 prior lines suggests that, in advanced MM, further genomic alterations may also be contributing to ixazomib sensitivity. Disclosures Di Bacco: Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Bahlis:BMS: Honoraria; Onyx: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau. Avet-Loiseau:Takeda: Speakers Bureau; Janssen: Speakers Bureau; Celgene: Speakers Bureau; Sanofi: Speakers Bureau. Masszi:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Ganly:Medipics: Equity Ownership; Novartis: Honoraria; Roche: Honoraria. Cavo:Bristol-Myers Squibb: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Langer:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Kumar:Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; Onyx: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Glycomimetics: Consultancy; Kesios: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding. Berg:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Lin:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Li:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Badola:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Shen:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Zhang:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Wang:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Esseltine:Takeda: Equity Ownership; Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment; Johnson and Johnson: Equity Ownership. Luptakova:Takeda Oncology: Employment. van de Velde:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Moreau:Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Amgen: Honoraria.

Abstract DNA methylation at cytosine position 5 (5mC) is a genetic modification that regulates gene expression and is critical for cancer initiation and development. The methylcytosine dioxygenase TET2 participates in active DNA demethylation by converting 5mC to 5-hydroxymethylcytosine (5hmC) and its subsequent intermediates thereby regulating gene expression. TET2 gene is frequently mutated in hematological disorders, including 15-25% of myelodysplastic syndrome (MDS) cases. Loss of TET2 function leads to DNA hypermethylation and subsequent dysregulated gene expression in hematopoietic stem cells, and has been considered as an initial step of myeloid malignant transformation including MDS and acute myeloid leukemia (AML). While enzymatic activity of TET2 is well studied, little is known about post-translational modifications (PTMs) that regulate its activity in hematopoietic cells. Lysine acetylation, one of the most crucial PTMs, occurs in a variety of proteins and modulates protein-protein or protein-nucleic acids interactions. Here, we first observed that ectopic expression of histone acetyltransferase EP300 or CREB binding protein (CBP) increased the endogenous TET2 acetylation in 293T cells. Mass spectrometry analysis of TET2 identified several conserved acetylated lysine residues located on the exterior surface of its catalytic domain (CD), which is predicted to be essential for the interaction of TET2 with DNA. Modeling of TET2/DNA interaction using x-ray crystal structure (PDB code: 4NM6) indicates acetylated TET2 interferes with DNA binding thereby impairing the catalytic activity of the enzyme. Like other acetylated proteins, TET2 modification is reversible, since ectopic expression of the protein deacetylase Sirtuin1 (SIRT1) significantly decreases acetylation of TET2. A domain mapping analysis revealed that SIRT1 preferentially interacts with TET2 CD, further supporting the regulatory role of SIRT1 on TET2. To explore the role of SIRT1 mediated deacetylaion of TET2 in more physiological condition, SIRT1 knockdown (KD) MDS-L cells as well as SIRT1 knockout (KO) murine hematopoietic c-kit+ cells were used. In MDS-L cells derived from a MDS patient, SIRT1 KD promoted hyperacetylation of endogenous TET2 which is associated with 50% decrease in 5hmC levels compared to control (p=0.03, n=3). Similarly, SIRT1 KO murine c-Kit+ cells showed TET2 hyperacetylation and 62% decrease in 5hmC compared to wildtype (WT) counterpart (p=0.009, n=5). Phenotypically, the transforming ability of SIRT1 shRNA transduced MDS-L cells was evaluated in vitro through serial replating assay. In contrast to first plating which showed no difference between two groups, the number of colonies derived from SIRT1 KD cells was 2.39 fold higher than that from the control shRNA transduced group after two rounds of replating (p=0.0002, n=3). Importantly, we tested the in-vivo effect of SIRT1 KD on MDS-L cells engrafted in immunodeficient NSGS mice. SIRT1 KD significantly enhanced MDS-L cell engraftment compared to control shRNA (50±5% of human CD45+ cells in SIRT1 KD group vs. 19±4% human CD45+ cells in control group, p=0.003, n=6). Interestingly, increased transformation capacity of SIRT1 KD MDS-L cells was associated with decreased expression of TET2 regulated tumor suppressor genes. For example, expression levels of Mtss1 and Dusp6 were significantly decreased in SIRT1 KD MDS-L cells compared to control shRNA counterpart (Mtss1, 49±5% decrease compared to control, p=0.005; Dusp6, 25±2% decrease compared to control, p=0.01). Similar results were observed in SIRT1 KO cells. The validation of physical binding of TET2 to its targets is ongoing. Moreover, the effect of an SIRT1 allosteric activator-SRT1720 in MDS-L cells is similar to that of Vitamin C, a known TET2 activator. Specifically, SRT1720 significantly increased Mtss1 expression in MDSL cells (p=0.0005, n=3), indicating that SIRT1 agonist may lead to activation of TET2 downstream tumor suppressor genes. In summary, our data demonstrate that TET2 activity can be functionally modified by acetylation and may be enhanced in MDS patients that do not harbor loss-of-function mutations through the SIRT1 mediated deacetylation. These results support further exploration of molecular mechanisms inducing TET2 acetylation, and evaluation of SIRT1 activation as a potential therapeutic approach in MDS. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: IL-15 has been used in clinical trials with PD-1 antibody to overcome cancer resistance to PD-1 monotherapy in cancer patients. However, it is not clear what T cell population would be expanded to mediate tumor control in IL-15 in combination with PD-1 antibody. We recently found CX3CR1+ cytotoxic CD8+ T cells, which increased in responders compared with non-responders after anti-PD-1 in melanoma patients, demonstrated an increased transcription of CD122 (IL2RB). Since CD122 is a subunit of IL-15 receptor, we hypothesized that IL-15 in combination with anti-PD-1 therapy may have the ability to expand CX3CR1+ cytotoxic CD8+ T cells for tumor control. Methods and findings: To examine whether IL-15 plus anti-PD-1 expand CX3CR1+ cytotoxic CD8+ T cells in tumor tissues, we treated tumor-bearing mice with IL-15/IL-15Rα complexes alone or with PD-1 antibody and measured the frequency of CX3CR1+Granzyme B+ CD8+ T cells. As only the combination therapy induced dramatic tumor regression, we found the highest increase of CX3CR1+Granzyme B+ CD8 T cells in tumor tissues in the group treated with IL-15 plus anti-PD-1 (28.87±3.19% vs. 7.91±1.32%, p&lt;0.01) compared with groups treated with either IL-15 alone or anti-PD-1 alone. In contrast to wild type mice, IL-15 plus anti-PD-1 did not suppress tumor growth in CX3CR1-KO mice. To determine whether IL-15 alone can also expand human CX3CR1+ cytotoxic CD8+ T cells, we cultured peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 5) with IL-15 for 24 hours in vitro. We found IL-15 (5 ng/mL) only modestly increased the frequency of CX3CR1+ Granzyme B+ CD8+ T cells than those cultured with no IL-15 (39.9±6.44% vs. 27.56±4.98%). To test whether this increase is due to enhanced proliferation of this subset of CD8+ T cells, we measured the expression of Ki-67, a cell proliferative marker, in CX3CR1+ CD8+ T cells by intranuclear staining. We found IL-15 modestly induced more proliferating CX3CR1+ CD8+ T cells in vitro culture compared with control group without IL-15 (6.25±0.54% vs. 0.91±0.25%). Summary: Taken together, we found IL-15 combined with anti-PD-1 therapy has the ability to significantly expand CX3CR1+ cytotoxic CD8+ T cells, however IL-15 alone may have limited ability to significantly expand this effector cell population. Our studies provide a new rational for combination therapy of IL-15 and PD-1 antibody to overcome cancer resistance for patients who did not respond to initial PD-1 therapy. Citation Format: Henan Zhang, Xin Liu, Siyu Cao, Lingling Chen, Susan Harrington, Ying Li, Aaron Mansfield, Sean Park, Yiyi Yan, Roxana Dronca, Haidong Dong. Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4080.

Abstract The metabolic signatures of cancer cells are often associated with elevated glycolysis which promotes alteration in cellular bioenergetics. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway has emerged as a potential anti-cancer target associated with many aspects of neoplastic progression. Pharmacological (PFK158 treatment) and genetic inhibition of PFKFB3 activity, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and inhibits cell proliferation in vitro by arresting the cells in the G0/G1 phase induced cell death in malignant pleural mesothelioma (MPM). It induces an early response of increased macropinocytosis and drastic endosomal vacuolization to overcome the nutrient stress both in sarcomatoid (H28) and epithelioid (EMMeso) MPM cells. Inhibition of PFKFB3 leads to the activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called “methuosis”. Transmission Electron Microscopy analysis showed that pharmacological and genetic inhibition of PFKFB3 activity leads to the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Confocal microscopy revealed F-actin and α-tubulin disassembly leading to the disruption of cell structure integrity. In parallel, PFKFB3 expression inversely modulates endoplasmic reticulum (ER) stress in MPM. Pharmacological and genetic inhibition of PFKFB3 led to an escalation in ER activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression levels. However, EHT1864, a Rac1 inhibitor, could not rescue MPM cells from ER stress, suggesting that the activation of macropinocytosis and ER stress are a result of two independent PFKFB3 modulated adaptive responses. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that the two crucial biomolecules of each pathway, Rac1 and Calnexin, interact with each other only at the later stages of PFKFB3 inhibition. Finally, PFK158, in a combination of with CBPt, demonstrated a significant reduction in tumor weight in mesothelin overexpressed EMMeso xenograft model. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM. Citation Format: Sayantani Sarkar Bhattacharya, Ling Jin, Debarshi Roy, Deokbeom Jung, Prabhu Thirusangu, Yinan Xiao, Julie Staub, Julian Molina, Viji Shridhar. PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2658.

Objectives: The incidence of shoulder and elbow overuse injury continues to rise in youth baseball players. Several throwing programs designed to reduce stress on the elbow have been described, but most are not evidence-based. The aim of this study was to compare the kinetics and kinematics between mound and flat-ground pitching at two different distances with the goal of developing evidenced-based injury prevention and recovery guidelines for youth throwers. Methods: Fifteen healthy, high school varsity-level baseball pitchers (mean age 16.7 ± 0.7 yrs; height 182.2 ± 6.2 cm; weight 76.0 ± 9.4 kg;) participated in the study. Players were fitted with a motusBASEBALLTM sensor and sleeve (Motus Global, Rockville Centre, NY), which has been shown to have good reliability and correlation with in-laboratory measures. Each pitcher was asked to pitch 5 fastballs to a catcher under each of the 4 conditions: mound at 60.5 ft (regulation distance), flat ground at 60.5 ft, mound at 50.5 ft, and flat ground at 50.5 ft. For each pitch, the sensor recorded arm speed, arm slot, shoulder rotation, and elbow varus torque. Ball velocity was tracked with a radar gun (Stalker Radar, Richardson, TX). Linear mixed-effects models were used to account for both within and between-subject variability. A multivariable model was used to evaluate the association of mound pitching (vs flat ground), distance (50.5 vs 60.5 ft), and their interaction on each of the following outcomes: arm speed, arm slot, shoulder rotation, elbow varus torque, and ball velocity. Results: There were no statistically significant effects of mound vs flat ground or distance variation on both arm speed or shoulder rotation. Arm slot was significantly higher on pitches from the mound at 60.5 ft [+4.58 (95% CI: 1.26, 7.90), p=0.007]. Elbow varus torque was significantly lower on throws from the mound [-1.88 (95% CI: -3.56, -0.20), p=0.03] and from a longer distance [-2.21 (95% CI: -3.89, -0.53), p=0.01]. Pitches thrown from the mound were significantly faster compared to flat ground at both distances, with throws at 60.5 ft of greater velocity than at 50.5 ft [+1.03 (95% CI: 0.66, 1.40), p<0.001]. Please see the Table for full results. Conclusion: The findings suggest that throwing from the mound may not be higher risk compared to flat ground, contrary to long-standing notions. The lower elbow varus torque and higher arm slot, which has previously been shown to be associated with reduced stress, from throwing from the mound may even indicate a protective effect. Compared to 50.5 ft, there was lower elbow varus torque and faster ball velocity at the longer distance, indicating that elbow stress and ball velocity may not correlate perfectly, and radar guns may not be an appropriate surrogate measure of elbow varus torque. A better understanding of the kinetic and kinematic implications of various throwing programs will allow for designing programs that are based on objective data to achieve the goal of preventing injuries in young baseball players. [Table: see text]

Abstract The diagnostic utility of somatic mutations in the context of cytopenias is unclear: clonal hematopoiesis can be found in healthy individuals, patients with aplastic anemia (AA), clonal cytopenia of undetermined significance (CCUS) and myelodysplastic syndrome (MDS). We examined a cohort of 207 well‐characterized cytopenic patients with a 640‐gene next generation sequencing (NGS) panel and compared its diagnostic utility with a “virtual” 41 gene panel. The TET2 , SF3B1 , ASXL1 , and TP53 were the most commonly mutated genes (frequency > 10%). Mutations in the 640‐gene panel show high sensitivity (98.3%) but low specificity (47.6%) for diagnosis of MDS. Notably, mutations of splicing factors and genes in the RAS pathway are relatively specific to MDS. Furthermore, high variant allele frequency (VAF) predicts MDS: when the VAF is set at 20%, the positive predictive value (PPV) for MDS is 95.9%, with a specificity of 95.3%. The presence of two or more somatic mutations with ≥10% VAF showed a PPV of 95.2%. While the “virtual” 41‐gene panel showed a mild decrease in sensitivity (95.7% vs 98.3%), 100% specificity was observed when either VAF was set at ≥20% (100% vs 95.3%), or two or more somatic mutations had VAFs ≥ 10%. Our study shows targeted gene panel sequencing improves the diagnostic approach and accuracy for unexplained cytopenia, with its high sensitivity and high PPV for MDS when applying VAF cutoffs. Furthermore, a 41‐gene panel was shown to have at least comparable performance characteristics to the large 640‐gene panel.

Abstract Background Understanding the relationship between clinical and patient-reported outcomes (PROs) will help support clinical care and future clinical trial design of novel therapies for focal segmental glomerulosclerosis (FSGS). Methods FSGS patients ≥8 years of age enrolled in the Nephrotic Syndrome Study Network completed Patient-Reported Outcomes Measurement Information System PRO measures of health-related quality of life (HRQoL) (children: global health, mobility, fatigue, pain interference, depression, anxiety, stress and peer relationships; adults: physical functioning, fatigue, pain interference, sleep impairment, mental health, depression, anxiety and social satisfaction) at baseline and during longitudinal follow-up for a maximum of 5 years. Linear mixed-effects models were used to determine which demographic, clinical and laboratory features were associated with PROs for each of the eight children and eight adults studied. Results There were 45 children and 114 adult FSGS patients enrolled that had at least one PRO assessment and 519 patient visits. Multivariable analyses among children found that edema was associated with global health (−7.6 points, P = 0.02) and mobility (−4.2, P = 0.02), the number of reported symptoms was associated with worse depression (−2.7 per symptom, P = 0.009) and anxiety (−2.3, P = 0.02) and the number of emergency room (ER) visits in the prior 6 months was associated with worse mobility (−2.8 per visit, P &lt; 0.001) and fatigue (−2.4, P = 0.03). Multivariable analyses among adults found the number of reported symptoms was associated with worse function in all eight PROMIS measures and the number of ER visits was associated with worse fatigue, pain interference, sleep impairment, depression, anxiety and social satisfaction. Laboratory markers of disease severity (i.e. proteinuria, estimated glomerular filtration rate and serum albumin) did not predict PRO in multivariable analyses, with the single exception of complete remission and better pain interference scores among children (+9.3, P = 0.03). Conclusions PROs provide important information about HRQoL for persons with FSGS that is not captured solely by the examination of laboratory-based markers of disease. However, it is critical that instruments capture the patient experience and FSGS clinical trials may benefit from a disease-specific instrument more sensitive to within-patient changes.