Infection is the most common cause of mortality among patients with multiple myeloma undergoing autologous stem-cell transplant. Understanding the risk factors that lead to higher probability of infection is instrumental in their prevention. Although some risk factors are known, there are no robust predictive models for infection among myeloma patients receiving a hematopoietic stem-cell transplant. In this research study, we compared a variety of machine learning algorithms to reveal clinical features pre-transplant that are most predictive of infection post-transplant. Using features extracted from the electronic health record by means of natural language processing and manual abstraction, we trained our model using patients diagnosed with multiple myeloma who received an autologous stem-cell transplant at the Seattle Cancer Care Alliance. Along with being robust to missing data, the model is capable of processing diverse, heterogeneous data and identifying a class of predictive factors that can guide treatment decisions. Preliminary results point to lab tests such as lymphocyte, neutrophil, and platelet counts as the strongest predictive features for infection, and further exploration is underway to understand the full impact of these and other features.

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue cancer. Currently, the standard treatment regimen for RMS patients remains relatively unchanged. Conventional treatment of RMS includes a combination of chemotherapy, radiation, and surgical tumor resection. Unfortunately, with the heterogeneity of cancer between patients, these therapies are not always effective and can cause undesired health issues for the patient due to their non-specific effects. Targeted drug therapy can help patients live more normal lives. The angiotensin II receptor type 1 (AGTR1) and 2 (AGTR2) are potential targeted therapy targets that can inhibit RMS cancer growth and cause less side effects compared to conventional therapies. AGTR1/2 are the main effectors in the renin angiotensin system regulating cardiovascular health. While there are Federal Drug Administration (FDA) drugs blocking these receptors to treat high blood pressure (Irbesartan), AGTR1 and AGTR2 have yet to be investigated for their role in RMS. Tumor propagating cells (TPC), which function as tumor stem cells in RMS, are proposed to drive tumor metastasis and relapse through a process called self-renewal. Preliminary disruption of AGTR1 and AGTR2 in the two major subtypes of RMS (embryonal and alveolar) with the CRISPR/Cas9 gene editing system resulted in decreased tumor cell growth and self-renewal capabilities. RMS cell lines treated with Irbesartan also decreased in viability compared to untreated cells. Based on our preliminary results, I propose that AGTR1/2 plays a role in regulating RMS cell growth and self-renewal. Further functional characterization of AGTR1/2 and investigation of the cellular mechanism by which AGTR1/2 regulates RMS tumor cell growth and self-renewal can provide a strong rationale for prioritizing AGTR1/2 as targets for drug therapies to slow the progression of RMS without greatly compromising more of the patient's health.

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric cancer that has poor prognosis for patients with relapsed disease. ERMS typically harbors mutations in one of 3 RAS proteins. Mutations in NRAS, a member of the RAS family, have been shown to be a driver for many different cancers, including ERMS. The Chen lab previously demonstrated that genetic disruption of the NRAS gene by the CRISPR/Cas9 technique successfully reduced ERMS tumor growth in a human xenograft mouse model. However, these mice also experienced disease relapse. I was able to confirm successful targeting of at least one copy of NRAS in ERMS cells. I have subsequently isolated clones of ERMS cells that continued to grow despite the presence of NRAS gene disruption. In my investigation of candidate genes and pathways that might be responsible for driving continued ERMS tumor cell growth, I saw an increase in the level of YAP1 being produced in NRAS-targeted ERMS cells when compared to the control cells. Based on my preliminary findings, once NRAS is successfully disrupted in ERMS cells, tumor relapse is then driven instead by YAP1. This study could provide novel insight into the mechanisms underlying cancer relapse in response to NRAS targeting and promise alternative treatment plans for ERMS patients.

Currently, there is no clinical best practice in place to support and manage follow up care for previvors, individuals who have tested positive for one or more mutations that increase cancer risk. Patients and providers need support in making complex decisions about follow-up. This project aims to design ‘previvor plans’, modeled after survivorship care plans received by cancer survivors, to help inform and support previvors after they receive a positive test result. Content of the previvor plans includes recommendations for screening, prophylactic surgery, pharmacological interventions, and lifestyle changes based on national guidelines as well as resources to receive further education and support. This project is a part of a larger study which evaluates two different methods of engaging patients in genetic testing for cancer risk across 12 different primary care clinics. Each patient in the study who tests positive for a mutation that increases cancer risk receives a previvor plan tailored with resources and recommendations specific to their test results and location. Use of the plans will be evaluated through surveys and interviews of previvor patients. If successful, the creation of previvor plans will provide a tool which can be used by patients and their providers to manage follow up care after receiving a positive test result.

Liver cancer is the world's second most deadly malignancy with a five-year survival rate of just 18%. Hepatocellular Carcinoma (HCC) accounts for most of the cases and its incidence is projected to rise to one million deaths per year by 2030. Unfortunately, HCC is extremely difficult to treat because several molecular pathways that promote drug resistance are upregulated in tumors, the most important of these being the epithelial-to-mesenchymal transition (EMT). Under physiological conditions the EMT regulates embryonic development, wound healing and tissue repair. However, cancer cells can hijack EMT signaling pathways to acquire a metastatic and drug resistant phenotype. Therefore, cell signaling enzymes that promote the cancer cell EMT are an attractive target for pharmacological intervention. Recently, we discovered that 71 protein kinases are highly enriched in mesenchymal HCC cells. To determine if these signaling enzymes are bona fide drivers of the EMT and drug resistance, we generated kinase RNAi knockout cell lines, quantified differences in EMT marker mRNA expression by qPCR, determined EMT pathway activation using quantitative proteomics, and tested differences in drug sensitivity. Here we demonstrate that inhibition of several of the 71 candidate EMT kinases reverses the phenotypic transition and sensitizes drug resistant HCC cells to chemotherapy. We conclude that these kinases could present attractive targets for the development of novel drugs that block cancer metastasis and overcome therapy resistance.

Fanconi Anemia (FA) is a recessively inherited genetic disorder resulting from a loss-of-function mutation in any of the 23 genes that comprise the Fanconi (FANC) gene family. FA is characterized by congenital abnormalities, bone marrow failure, and a predisposition to hematologic and solid cancers. The elevated risk of cancer in FA is largely limited to the squamous mucosa lining of the oropharynx, esophagus and vulva, and is not ameliorated by successful prior bone marrow transplantation. Apart from surgery, effective treatment of these cancers is limited by patient hypersensitivity to standard-of-care therapies that include ionizing radiation and DNA cross-linking drugs. The pathogenesis of FA-derived cancers is still not well understood. As a result, we are developing a “Fanconi Anemia Cancer Cell Line Resource” to provide preclinical models for research on FA-derived head and neck squamous cell carcinoma. The resource will include genomically well-defined, isogenic HNSCC cell line pairs (FA-patient/complemented cells) from patients with FA and isogenic cell line trios (wild-type/FANCA-deficient/FANCA-complemented cells) from control sporadic HNSCC cells. FANCA-deficient control cells were generated by targeted Cas9-mediated deletions in both FANCA alleles. Complementation of the resulting FANCA-deficient cells and patient cells was accomplished by targeted FANC- transgene insertion at a chromosome 4 ‘safe harbor site’. The molecular and cellular phenotype of the clonally-derived knockout and knockout/complemented cell lines was verified by PCR analyses; by Western blot and Multiple Reaction Monitoring (MRM) mass spectrometry analysis of FANCA and FANCD₂ protein expression/modification ± DNA damage; and by cell survival as a function of mitomycin-C (MMC) dose. The FA-HNSCC and sporadic HNSCC cell lines will be available for investigators via the FARF- sponsored Fanconi Anemia Cancer Cell Line Repository. We anticipate this versatile resource will be valuable in the development of novel treatment and faciliate in improving our understanding of FA cancers.
 

Chronic myeloid leukemia is among myeloproliferative neoplasms that cause an increase of myeloid cells. All CML cases have a reciprocal translocation causing the novel fusion BCR-ABL gene causing the disease. BCR-ABL protein is a drug target and BCR-ABL mRNA is a diagnostic target. The polymerase chain reaction (PCR) is a technique used to diagnose and monitor treatment response in CML. Tyrosine kinase inhibitors (TKIs) have raised the survival rate of CML patients from an average lifespan of 6 years to a normal lifespan. However, monitoring and diagnosing CML patients requires expensive equipment and technical expertise. Medical testing and therapy of CML in lower to middle income countries is difficult, because drugs are expensive and access to sophistical equipment rare. However, when the disease is confirmed by testing, a non-profit organization (the Max Foundation) can provide TKIs free for life through partnerships with pharmaceutical companies. Dried blood spots (DBS) can potential stabilize nucleic acid for long periods of time. This approach has facilitated retrieval of the genetic material on the DBS that can be used to analyze more genetic analysis related to CML response and progression and has reduced transportation costs. In this study, we isolated DNA & RNA from DBS, quantified the genetic material, performed quality control, and carried out DNA & RNA sequencing-based on gene mutation assays. Tapestation and qPCR were performed to check the quality of the genetic material. DNA samples showed a higher DNA integrity number with an average of 7.7. However, the RNA samples demonstrated a lower RNA integrity number with an average of 1.8. The DNA samples had excellent quality whereas the RNA samples had poor quality but were acceptable enough to carry on with our study. These results will help us determine if genetic material on DBS can be used for more genetic analysis.

Nicotine is the addictive substance found in various tobacco products. CYP2A13 is an enzyme localized in the lungs, metabolizes tobacco-specific nitrosamine carcinogens that contribute to lung cancer. Therefore, pinpointing CYP2A13 inhibitors is an approach to lower tobacco-based lung cancer risk. Cinnamaldehyde is a common flavoring agent in the fluids of electronic nicotine vaping devices. Cinnamaldehyde was found to be a potent inhibitor of CYP2A6, another enzyme that metabolizes nicotine. Because CYP2A13 and CYP2A6 exhibit overlap in substrate/inhibitor selectivity, the goal here was to evaluate the inhibition of CYP2A13 by cinnamaldehyde. A time-dependent inhibition coumarin assay was performed to determine the kinetic parameters for cinnamaldehyde in recombinant CYP2A13. Primary incubations contained cinnamaldehyde, CYP2A13 Supersomes, and potassium phosphate buffer. Incubations were initiated with NADPH. Secondary incubations contained coumarin, NADPH, and potassium phosphate buffer. At selected time points, an aliquot of the primary incubation mixture was transferred to the secondary incubation tubes, which were terminated with trichloroacetic acid after heating at 37°C for 5.5 minutes. A linearity study was conducted to determine the appropriate termination time. CYP2A13 activity was measured by the detection of hydroxycoumarin using high-performance liquid chromatography (HPLC) and a fluorescence detector. Hydroxycoumarin formation decreased with time and inhibitor concentrations. Maximal inhibition following an 18-minute incubation was 38.3 ± 1.6 and 4.0 ± 0.6%. The maximal rate of inhibition was 0.109 per minute. The results provide evidence that cinnamaldehyde is a time-dependent inhibitor of CYP2A13. Furthermore, cinnamaldehyde appears to be a more potent inhibitor of CYP2A13 than CYP2A6, based on the maximal rate of inhibition. The results imply that cinnamaldehyde could interfere with the bioactivation of nitrosamine lung carcinogens. Additional kinetic studies are needed to confirm the results of this study and to evaluate the safety and toxicity profiles of cinnamaldehyde in complex physiological models.