Prospective comparison of risk factors for firearm suicide and non-firearm suicide in a large population-based cohort of current and former US service members: findings from the Millennium Cohort Study.

Background: Suicide is a leading cause of death among service members and veterans. Among suicide methods, firearms are the most lethal and commonly used method among military populations. Limited research has compared risk factors for the various suicide methods. This study evaluated and compared risk factors for firearm versus non-firearm suicides using data from the Millennium Cohort Study, a large longitudinal military cohort. Methods: Using a competing risk approach, we identified factors associated with each suicide method. Risk factors included demographics, mental health diagnoses, mental health symptoms, military-specific characteristics, health behaviors, and psychosocial factors. Cause of death was assessed from July 1, 2001, through December 31, 2018. Findings: Among 201,565 eligible participants with a mean [SD] age of 29.0 [58.1] years, there were 139,789 (69.3%) male, 61,776 (30.7%) female, 15,927 (7.9%) Hispanic, 24,667 (12.3%) non-Hispanic Black, 14,138 (7.0%) Asian, Pacific Islander, American Indian or Multiracial, and 146,736 (72.8%) non-Hispanic White participants. During the study period, 330 died by firearm suicide and 168 died by non-firearm suicide. Overall, effect estimates for risk factors were similar across both methods of suicide. After adjustment, men (HR: 3.69, 95% CI: 2.59, 5.24) and those who screened positive for depression (HR: 1.97, 95% CI: 1.36, 2.87) had an elevated risk for firearm suicide. In contrast, those who self-reported a history of bipolar diagnosis (HR: 3.40, 95% CI: 1.76, 6.55) had significantly increased risk for non-firearm suicide. Interpretation: Findings suggest that prevention and intervention strategies overall may not need to be differentiated by specific demographic, military, or health factors. Targeted interventions that consider sex and mental health screens might have relative utility in preventing firearm related suicide risk compared with non-firearm suicide. Funding: Military Operational Medicine Research Program, Defense Health Program, and Department of Veterans Affairs.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Cervical cancer screening compliance among active duty service members in the US military.

Previous research suggests active duty service members (ADSM) experience higher rates of human papilloma virus infection and cervical dysplasia, which puts them at greater risk for cervical cancer. The current study examined crude rates and correlates of cervical cancer screening compliance in 2003-2015 among screening-eligible ADSM in the Millennium Cohort Study (MCS). Data were drawn from the MCS, Defense Manpower Data Center, and Military Health System Data Repository. Screening eligibility and compliance were calculated each year and initial analyses examined crude rates of compliance. Generalized estimating equations were calculated to determine whether sociodemographic, military, and mental/behavioral health covariates were associated with cervical cancer screening compliance. A majority of participants were 21-29 years old (79.4%), non-Hispanic White (60.6%), and enlisted (82.2%). Crude rates of cervical cancer screening compliance increased from 2003 (61.2%) to 2010 (83.1%), and then declined from 2010 to 2015 (59.8%). Older ADSM and those who had a history of deployment had lower odds of screening compliance. ADSM in the Air Force and those in healthcare occupations had higher odds of screening compliance. Study findings suggest that cervical cancer screening compliance is declining among ADSM. Interventions to improve screening should target groups with lower screening compliance.

Factors Associated with Human Papillomavirus Vaccine Initiation and Compliance Among U.S. Military Service Members.

INTRODUCTION: The primary objective of the current study was to assess factors associated with Human Papillomavirus (HPV) vaccine initiation and compliance in a cohort of active duty US military service members (SM). MATERIALS AND METHODS: We included active-duty participants aged 18-26 years from the Millennium Cohort Study, a longitudinal cohort study of over 200,000 military SMs. The eligible study population included 22,387 female SMs and 31,705 male SMs. Vaccination was assessed over the period 2006-2017. Logistic regression was used to estimate the odds of vaccine initiation and compliance (3 doses within a 1-year period) in relation to demographic, military, health, and behavioral characteristics. RESULTS: Among female SMs, 37.8% initiated the vaccine and 40.2% of initiators completed the series within a year. Among male SMs, 3.9% initiated the vaccine and 22.1% of initiators completed the series within a year. Differences by sociodemographic factors, deployment status, branch of service, occupation, and smoking status-but not by selected mental health conditions-were observed. CONCLUSION: HPV vaccination uptake is subpar across all military service branches. Certain subgroups of SMs could be targeted to increase overall HPV vaccine coverage in the US military population.

Genetic ablation of Pim1 or pharmacologic inhibition with TP-3654 ameliorates myelofibrosis in murine models.

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasm (MPN). The JAK inhibitor Ruxolitinib can reduce constitutional symptoms but it does not substantially improve bone marrow fibrosis. Pim1 expression is significantly elevated in MPN/MF hematopoietic progenitors. Here, we show that genetic ablation of Pim1 blocked the development of myelofibrosis induced by Jak2V617F and MPLW515L. Pharmacologic inhibition of Pim1 with a second-generation Pim kinase inhibitor TP-3654 significantly reduced leukocytosis and splenomegaly, and attenuated bone marrow fibrosis in Jak2V617F and MPLW515L mouse models of MF. Combined treatment of TP-3654 and Ruxolitinib resulted in greater reduction of spleen size, normalization of blood leukocyte counts and abrogation of bone marrow fibrosis in murine models of MF. TP-3654 treatment also preferentially inhibited Jak2V617F mutant hematopoietic progenitors in mice. Mechanistically, we show that TP-3654 treatment significantly inhibits mTORC1, MYC and TGF-ß signaling in Jak2V617F mutant hematopoietic cells and diminishes the expression of fibrotic markers in the bone marrow. Collectively, our results suggest that Pim1 plays an important role in the pathogenesis of MF, and inhibition of Pim1 with TP-3654 might be useful for treatment of MF.

AXL Inhibitor TP-0903 Reduces Metastasis and Therapy Resistance in Pancreatic Cancer.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth.

TNK1 is a non-receptor tyrosine kinase with poorly understood biological function and regulation. Here, we identify TNK1 dependencies in primary human cancers. We also discover a MARK-mediated phosphorylation on TNK1 at S502 that promotes an interaction between TNK1 and 14-3-3, which sequesters TNK1 and inhibits its kinase activity. Conversely, the release of TNK1 from 14-3-3 allows TNK1 to cluster in ubiquitin-rich puncta and become active. Active TNK1 induces growth factor-independent proliferation of lymphoid cells in cell culture and mouse models. One unusual feature of TNK1 is a ubiquitin-association domain (UBA) on its C-terminus. Here, we characterize the TNK1 UBA, which has high affinity for poly-ubiquitin. Point mutations that disrupt ubiquitin binding inhibit TNK1 activity. These data suggest a mechanism in which TNK1 toggles between 14-3-3-bound (inactive) and ubiquitin-bound (active) states. Finally, we identify a TNK1 inhibitor, TP-5801, which shows nanomolar potency against TNK1-transformed cells and suppresses tumor growth in vivo.

Comparison of Posttraumatic Stress Disorder Checklist Instruments From Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition vs Fifth Edition in a Large Cohort of US Military Service Members and Veterans.

Importance: The definition of posttraumatic stress disorder (PTSD) changed markedly between the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) and DSM-5, creating challenges for studies and in medical settings spanning this transition. Objective: To evaluate the ability to compare and assess PTSD, based on DSM-IV and DSM-5 criteria, using PTSD Checklists (PCLs). Design, Setting, and Participants: This diagnostic study was conducted with survey data collected in October 2019, from the Millennium Cohort Study, a population-based US military cohort study. The population for the present study was restricted to a subset of initial web responders of the 2019 survey cycle, randomly assigned to 1 of 4 survey groups. Exposures: Each group received the DSM-IV and DSM-5 PCL (PCL-Civilian [PCL-C] version and PCL for DSM-5 [PCL-5]). PCL instruments were counterbalanced to control for order effects. Main Outcomes and Measures: Survey data were used to assess PTSD (using the PCL-C and PCL-5), major depressive disorder (using the Patient Health Questionnaire), generalized anxiety (using the Generalized Anxiety Disorder scale), and problem drinking (using the Patient Health Questionnaire). Demographic and military characteristics included age, sex, race/ethnicity, marital status, education, service branch, pay grade, enrollment panel, and military service status. Results: Among the 1921 participants (mean [SD] age, 50.1 [12.5] years), 1358 (70.7%) were men, 1638 (85.3%) were non-Hispanic White individuals, 1440 (75.0%) were married, and 1190 (61.9%) had at least a bachelor's degree; 295 (15.4%) had probable PTSD according to DSM-IV criteria with PCL-C compared with 286 (14.9%) using DSM-5 criteria with PCL-5 (κ = 0.77). There was substantial agreement between PCLs for probable PTSD based on DSM-IV criteria (295 [15.4%] with PCL-C; 316 [16.4%] with PCL-5; κ = 0.80) and DSM-5 criteria (286 [14.9%] with PCL-5; 258 [13.4%] with PCL-C; κ = 0.77). Estimated PTSD sum scores showed excellent agreement with observed scores. Using an established crosswalk, PCL-5 sum scores estimated with the PCL-C were similar to observed PCL-5 scores. Of the 17 corresponding items between the 2 instruments, 16 had substantial agreement. Appending 2 additional PCL-C items to the PCL-5 did not significantly alter estimates. The PCL-C and PCL-5 had nearly identical associations with comorbid conditions. Conclusions and Relevance: The findings of this diagnostic study suggest that PTSD can be successfully assessed and compared over time with either PCL instrument in veteran and military populations.

Mental health, physical health, and health-related behaviors of U.S. Army Special Forces.

OBJECTIVES: To prospectively examine the health and health-related behaviors of Army Special Forces personnel in comparison with two distinct, but functionally similar Army groups. METHODS: Special Forces, Ranger Qualified, and General Purposes Forces enrolled in the Millennium Cohort Study were identified using data from the Defense Manpower Data Center. Using prospective survey data (2001-2014), we estimated the association of Army specialization with mental health, social support, physical health, and health-related behaviors with multivariable regression models. RESULTS: Among the 5,392 eligible participants (84.4% General Purposes Forces, 10.0% Special Forces, 5.6% Ranger Qualified), Special Forces personnel reported the lowest prevalence of mental disorders, physical health problems, and unhealthy behaviors. In the multivariable models, Special Forces personnel were less likely to report mental health problems, multiple somatic symptoms, and unhealthy behaviors compared with General Purpose Forces infantrymen (odds ratios [OR]: 0.20-0.54, p-values < .01). Overall, Special Forces personnel were similar in terms of mental and physical health compared with Ranger Qualified infantrymen, but were less likely to sleep < 5 hours/night (OR: 0.60, 95% confidence intervals: 0.40, 0.92) and have 5 or more multiple somatic symptoms (OR: 0.69, 95% CI: 0.49, 0.98). Both Special Forces personnel and Ranger Qualified infantrymen engaged in more healthy behaviors compared with General Purpose Forces infantrymen (OR: 2.57-6.22, p-values<0.05). Engagement in more healthy behaviors reduced the odds of subsequent adverse health outcomes, regardless of specialization. CONCLUSIONS: Army Special Forces personnel were found to be mentally and physically healthier than General Purpose Forces infantrymen, which may in part be due to their tendency to engage in healthy behaviors. Findings indicate that engagement in a greater number of healthy behaviors may reduce odds for subsequent adverse outcomes.

Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFß signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFß, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFß, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Ecological Momentary Assessment of Factors Associated with Water Intake among Adolescents with Kidney Stone Disease.

PURPOSE: Maintaining high water intake decreases kidney stone recurrence but is difficult to do. Strategies to reduce stone recurrence among adolescents are lacking. We conducted an ecological momentary assessment study to identify factors associated with water intake in adolescents with nephrolithiasis. MATERIALS AND METHODS: The study population consisted of 15 female and 10 male patients 12 to 18 years old with at least 1 prior kidney stone. For 7 days participants used "smart" bottles to self-monitor water intake and received questionnaires randomly 4 times daily, which were completed in real time on mobile devices. The questionnaires ascertained awareness of water intake volume, awareness of water intake goals, perceived need to drink, access to water, alternative beverage consumption and attitudes toward bathrooms. Linear mixed effects models were fit to estimate the association between momentary responses and daily water intake. RESULTS: During 175 person-days 595 assessments (85%) were completed. Median daily water intake was 1,304 ml (IQR 848-1,832) and 20% of participants met their intake goal for 4 days or more. Unawareness of water intake volume was associated with drinking 690 ml less water per day (p = 0.04). A strong self-perceived need to drink more was associated with drinking 1,954 ml less water each day compared to no self-perceived need to drink more (p <0.01). Unawareness of intake goals was weakly associated with drinking 1,129 ml less water each day (p = 0.1). Access to water, alternative beverage consumption and bathroom aversion were not associated with water intake. CONCLUSIONS: Unawareness of water volume consumed and low responsiveness to perceived need to drink more were associated with low water intake. Interventions that help adolescents recognize when and identify how to increase water intake may be effective in decreasing stone recurrence.

A Dynamic Graph Cuts Method with Integrated Multiple Feature Maps for Segmenting Kidneys in 2D Ultrasound Images.

RATIONALE AND OBJECTIVES: Automatic segmentation of kidneys in ultrasound (US) images remains a challenging task because of high speckle noise, low contrast, and large appearance variations of kidneys in US images. Because texture features may improve the US image segmentation performance, we propose a novel graph cuts method to segment kidney in US images by integrating image intensity information and texture feature maps. MATERIALS AND METHODS: We develop a new graph cuts-based method to segment kidney US images by integrating original image intensity information and texture feature maps extracted using Gabor filters. To handle large appearance variation within kidney images and improve computational efficiency, we build a graph of image pixels close to kidney boundary instead of building a graph of the whole image. To make the kidney segmentation robust to weak boundaries, we adopt localized regional information to measure similarity between image pixels for computing edge weights to build the graph of image pixels. The localized graph is dynamically updated and the graph cuts-based segmentation iteratively progresses until convergence. Our method has been evaluated based on kidney US images of 85 subjects. The imaging data of 20 randomly selected subjects were used as training data to tune parameters of the image segmentation method, and the remaining data were used as testing data for validation. RESULTS: Experiment results demonstrated that the proposed method obtained promising segmentation results for bilateral kidneys (average Dice index = 0.9446, average mean distance = 2.2551, average specificity = 0.9971, average accuracy = 0.9919), better than other methods under comparison (P < .05, paired Wilcoxon rank sum tests). CONCLUSIONS: The proposed method achieved promising performance for segmenting kidneys in two-dimensional US images, better than segmentation methods built on any single channel of image information. This method will facilitate extraction of kidney characteristics that may predict important clinical outcomes such as progression of chronic kidney disease.

Assessment of the combination of temperature and relative humidity on kidney stone presentations.

Temperature and relative humidity have opposing effects on evaporative water loss, the likely mediator of the temperature-dependence of nephrolithiasis. However, prior studies considered only dry-bulb temperatures when estimating the temperature-dependence of nephrolithiasis. We used distributed lag non-linear models and repeated 10-fold cross-validation to determine the daily temperature metric and corresponding adjustment for relative humidity that most accurately predicted kidney stone presentations during hot and cold periods in South Carolina from 1997 to 2015. We examined three metrics for wet-bulb temperatures and heat index, both of which measure the combination of temperature and humidity, and for dry-bulb temperatures: (1) daytime mean temperature; (2) 24-h mean temperature; and (3) most extreme 24-h temperature. For models using dry-bulb temperatures, we considered four treatments of relative humidity. Among 188,531 patients who presented with kidney stones, 24-h wet bulb temperature best predicted kidney stone presentation during summer. Mean cross-validated residuals were generally lower in summer for wet-bulb temperatures and heat index than the corresponding dry-bulb temperature metric, regardless of type of adjustment for relative humidity. Those dry-bulb models that additionally adjusted for relative humidity had higher mean residuals than other temperature metrics. The relative risk of kidney stone presentations at the 99th percentile of each temperature metric compared to the respective median temperature in summer months differed by temperature metric and relative humidity adjustment, and ranged from an excess risk of 8-14%. All metrics performed similarly in winter. The combination of temperature and relative humidity determine the risk of kidney stone presentations, particularly during periods of high heat and humidity. These results suggest that metrics that measure moist heat stress should be used to estimate the temperature-dependence of kidney stone presentations, but that the particular metric is relatively unimportant.

Chronic lymphocytic leukemia cells from ibrutinib treated patients are sensitive to Axl receptor tyrosine kinase inhibitor therapy.

Earlier we have shown the expression of a constitutively active receptor tyrosine kinase Axl in CLL B-cells from previously untreated CLL patients, and that Axl inhibitor TP-0903 induces robust leukemic B-cell death. To explore whether Axl is an effective target in relapsed/refractory CLL patients, we analyzed CLL B-cells obtained from CLL patients on ibrutinib therapy. Ibrutinib-exposed CLL B-cells were treated with increasing doses (0.01- 0.50µM) of a new formulation of high-affinity Axl inhibitor, TP-0903 (tartrate salt), for 24 hours and LD50 doses were determined. Sensitivity of CLL B-cells was compared with known prognostic factors and effect of TP-0903 was also evaluated on Axl signaling pathway in CLL B-cells from this cohort. We detected sustained overexpression of Axl in CLL B-cells from CLL patients on ibrutinib treatment, suggests targeting Axl could be a promising strategy to overcome drug resistance and killing of CLL B-cells in these patients. We found that CLL B-cells from sixty-nine percent of relapsed CLL patients actively on ibrutinib therapy were found to be highly sensitive to TP-0903 with induction of apoptosis at nanomolar doses (≤0.50 µM). TP-0903 treatment effectively inhibited Axl phosphorylation and reduced expression levels of anti-apoptotic proteins (Mcl-1, XIAP) in ibrutinib exposed CLL B-cells. In total, our in vitro preclinical studies showing that TP-0903 is very effective at inducing apoptosis in CLL B-cells obtained from ibrutinib-exposed patients supports further testing of this drug in relapsed/refractory CLL.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors.

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.