The clinical manifestations and molecular pathogenesis of radiation fibrosis.

Advances in radiation techniques have enabled the precise delivery of higher doses of radiotherapy to tumours, while sparing surrounding healthy tissues. Consequently, the incidence of radiation toxicities has declined, and will likely continue to improve as radiotherapy further evolves. Nonetheless, ionizing radiation elicits tissue-specific toxicities that gradually develop into radiation-induced fibrosis, a common long-term side-effect of radiotherapy. Radiation fibrosis is characterized by an aberrant wound repair process, which promotes the deposition of extensive scar tissue, clinically manifesting as a loss of elasticity, tissue thickening, and organ-specific functional consequences. In addition to improving the existing technologies and guidelines directing the administration of radiotherapy, understanding the pathogenesis underlying radiation fibrosis is essential for the success of cancer treatments. This review integrates the principles for radiotherapy dosimetry to minimize off-target effects, the tissue-specific clinical manifestations, the key cellular and molecular drivers of radiation fibrosis, and emerging therapeutic opportunities for both prevention and treatment.

CD8+ Tumor-Infiltrating Lymphocyte Abundance Is a Positive Prognostic Indicator in Nasopharyngeal Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC). EXPERIMENTAL DESIGN: Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50). RESULTS: In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03). CONCLUSIONS: Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.

Development and Validation of a Risk Model for Breast Cancer-Related Lymphedema.

Importance: Approximately 1 in 5 patients with breast cancer who undergo axillary lymph node dissection will develop lymphedema. To appropriately triage and monitor these patients for timely diagnosis and treatment, robust risk models are required. Objective: To evaluate the prognostic value of mammographic breast density in estimating lymphedema severity. Design, Setting, and Participants: This prognostic study collected data from July 16, 2018, to March 3, 2020, from the electronic health records of patients of the Cancer Rehabilitation and Survivorship Program at the Princess Margaret Cancer Centre in Toronto, Ontario, Canada. Participants included women who had completed curative treatment for a first diagnosis of breast cancer and who were referred to the program. Also included were a sample of patients in the general breast oncology population who were receiving follow-up care at the center during the same period but who were not referred to the program. All patients attended follow-up appointments at the Princess Margaret Cancer Centre from January 1, 2016, to May 1, 2018. The cohort was randomly split 2:1 to group patients into a training cohort and a validation cohort. Exposures: Participant demographic and clinical characteristics included age, sex, body mass index (BMI), medical history, cancer characteristics, and cancer treatment. Main Outcomes and Measures: Spearman correlation coefficient between measured and predicted volume of lymphedema was calculated. Area under the curve (AUC) values were generated for predicting the occurrence of at least mild lymphedema (volume, >200 mL) and severe lymphedema (volume, >500 mL) at the time of initial lymphedema diagnosis. Results: A total of 373 female patients (median [interquartile range] age, 52.3 [45.9-60.1] years) were eligible for this analysis. Multivariate linear regression identified 3 patient factors (age, BMI, and mammographic breast density), 1 cancer factor (number of pathological lymph nodes), and 1 treatment factor (axillary lymph node dissection) as independent prognostic variables. In validation testing, Spearman correlation revealed a statistically significant moderate correlation (coefficient, 0.42; 95% CI, 0.26-0.56; P < .001) between measured volume and predicted volume of lymphedema. The AUC values were 0.72 (95% CI, 0.60-0.83) for predicting the occurrence of mild lymphedema and 0.83 (95% CI, 0.74-0.93) for severe lymphedema. Conclusions and Relevance: This prognostic study found that patients with low breast density appeared to be at a higher risk of developing severe lymphedema. The finding suggests that by combining breast density with established risk factors a multivariate linear regression model could be used to predict the development of lymphedema and provide volumetric estimates of lymphedema severity in patients with breast cancer.

Inflammatory Biomarkers, Hematopoietic Stem Cells, and Symptoms in Breast Cancer Patients Undergoing Adjuvant Radiation Therapy.

BACKGROUND: Fatigue and insomnia are common symptoms experienced by breast cancer patients undergoing adjuvant radiation therapy (RT), yet the underlying mechanisms of these symptoms are unclear. In particular, the roles of hematopoietic stem cells (HSCs) and inflammatory cytokines remain to be elucidated. METHODS: Breast cancer patients (n = 147) completed questionnaires to longitudinally assess symptoms before, during, and after adjuvant RT. Phlebotomies were performed prior to RT, at the second and fifth treatment fractions, end of treatment (EOT), and 1 month after completing RT, assessing for CD34+, CD45+, full hematology, and 17 inflammatory cytokines. The associations between symptoms and all biomarkers were evaluated. All statistical tests were 2-sided. RESULTS: General fatigue and insomnia worsened with RT, with peak levels observed at EOT, which remained statistically significant even after controlling for anxiety and depression (P < .05 for all). CD34+, CD45+, white blood cell, and lymphocyte counts decreased, with the lowest levels also observed at EOT (P < .001). Fatigue and insomnia were associated with changes in both interferon γ-induced protein 10 (IP-10) - (P = .03 and P = .01, respectively) and tumor necrosis factor receptor II (TNF-RII) (P = .02 and P = .006, respectively), while mental fatigue was associated with increased matrix metalloproteinases-2 (MMP-2) levels (P = .03). Patients who received prior chemotherapy demonstrated statistically significantly greater severity in all symptoms, with lower baseline HSC levels. CONCLUSIONS: This is the first longitudinal study to examine linkages between symptoms, HSCs, and cytokines, demonstrating that fatigue and insomnia shared associations with increasing serum levels of IP-10 and TNF-RII, and mental fatigue was associated with increasing serum levels of MMP-2. Our findings highlight opportunities for further research into mechanisms and potential interventions for these symptoms.

MiR-34c downregulation leads to SOX4 overexpression and cisplatin resistance in nasopharyngeal carcinoma.

BACKGROUND: A major cause of disease-related death in nasopharyngeal carcinoma (NPC) is the development of distant metastasis (DM) despite combination chemoradiotherapy treatment. We previously identified and validated a four microRNA (miRNA) signature that is prognostic for DM. In this study, characterization of a key component of this signature, miR-34c, revealed its role in chemotherapy resistance. METHODS: Two hundred forty-six NPC patient biopsy samples were subject to comprehensive miRNA profiling and immunohistochemistry (IHC). Two human normal nasopharyngeal cell lines (immortalized; NP69 and NP460), as well as the NPC cell line C666-1, were used for miR-34c gain-of-function and loss-of-function experiments. Signaling pathways were assessed using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability was measured using the ATPlite assay. RESULTS: MiR-34c was downregulated in NPC patient samples, and confirmed in vitro to directly target SOX4, a master regulator of epithelial-to-mesenchymal transition (EMT). MiR-34c downregulation triggered EMT-representative changes in NP69 and NP460 whereby Snail, ZEB1, CDH2, and SOX2 were upregulated, while Claudin-1 and CDH1 were downregulated. Phenotypically, inhibition of miR-34c led to cisplatin resistance, whereas miR-34c over-expression sensitized NPC cells to cisplatin. TGFß1 decreased miR-34c and increased SOX4 expression in vitro. The TGFß receptor 1 inhibitor SB431542 reduced SOX4 expression and increased cisplatin sensitivity. Finally, IHC revealed that lower SOX4 expression was associated with improved overall survival in chemotherapy-treated NPC patients. CONCLUSION: miR-34c is downregulated in NPC. Repression of miR-34c was shown to increase SOX4 expression, which leads to cisplatin resistance, while TGFß1 was found to repress miR-34c expression. Taken together, our study demonstrates that inhibition of the TGFß1 pathway could be a strategy to restore cisplatin sensitivity in NPC.

hsa­miR­24 suppresses metastasis in nasopharyngeal carcinoma by regulating the c­Myc/epithelial­mesenchymal transition axis.

Distant metastasis is the major contributor to treatment failure and mortality in patients with nasopharyngeal carcinoma (NPC). The lack of effective treatment strategies for metastatic NPC is the major cause for the low survival rate. Therefore, it is crucial to understand the molecular mechanisms underlying NPC metastasis and to identify potential biomarkers for targeted therapy. MicroRNA (miRNAs or miRs) have been shown to play an important role in tumorigenesis and metastasis. In the present study, we aimed to evaluate the significance of hsa­miR­24 in NPC metastasis. Significantly lower hsa­miR­24 levels were observed in NPC metastatic tumors and higher hsa­miR­24 levels were associated with longer progression­free and metastasis­free survival durations. hsa­miR­24 overexpression inhibited cell proliferation, invasion and migration. Using bioinformatics approaches together with functional luciferase reporter assays, we demonstrated that the c­Myc 3'­UTR was a direct target of hsa­miR­24 in regulating NPC metastasis. Protein profiling analysis revealed that a high c­Myc expression was inversely associated with metastasis­free overall survival and with epithelial­mesenchymal transition (EMT). Furthermore, the overexpression of hsa­miR­24 decreased NPC cell invasive ability induced by the overexpression of c­Myc, associated with EMT epithelial marker (E­cadherin) restoration. Thus, on the whole, the findings of this study demonstrate that hsa­miR­24 suppresses metastasis in NPC by regulating the c­Myc/EMT axis, suggesting that hsa­miR­24 may be used as a prognostic factor and as a novel target for the prevention of NPC metastasis.

Dysregulation of the MiR-449b target TGFBI alters the TGFß pathway to induce cisplatin resistance in nasopharyngeal carcinoma.

Despite the improvement in locoregional control of nasopharyngeal carcinoma (NPC), distant metastasis (DM), and chemoresistance persist as major causes of mortality. This study identified a novel role for miR-449b, an overexpressed gene in a validated four-miRNA signature for NPC DM, leading to chemoresistance via the direct targeting of transforming growth factor beta-induced (TGFBI). In vitro shRNA-mediated downregulation of TGFBI induced phosphorylation of PTEN and AKT, increasing cisplatin resistance. Conversely, the overexpression of TGFBI sensitized the NPC cells to cisplatin. In NPC patients treated with concurrent chemoradiotherapy (CRT), the overall survival (OS) was significantly inversely correlated with miR-449b, and directly correlated with both TGFBI mRNA and protein expression, as assessed by RNA sequencing and immunohistochemistry (IHC). Mechanistically, co-immunoprecipitation demonstrated that TGFBI competes with pro-TGFß1 for integrin receptor binding. Decreased TGFBI led to increased pro-TGFß1 activation and TGFß1 canonical/noncanonical pathway-induced cisplatin resistance. Thus, overexpression of miR-449b decreases TGFBI, thereby altering the balance between TGFBI and pro-TGFß1, revealing a novel mechanism of chemoresistance in NPC.

Radiomic Biomarkers to Refine Risk Models for Distant Metastasis in HPV-related Oropharyngeal Carcinoma.

PURPOSE: Distant metastasis (DM) is the main cause of death for patients with human papillomavirus (HPV)-related oropharyngeal cancers (OPCs); yet, there are few reliable predictors of DM in this disease. The role of quantitative imaging (ie, radiomic) analysis was examined to determine whether there are primary tumor features discernible on imaging studies that are associated with a higher risk of DM developing. METHODS AND MATERIALS: Radiation therapy planning computed tomography scans were retrieved for all nonmetastatic p16-positive OPC patients treated with radiation therapy or chemoradiation therapy at a single institution between 2005 and 2010. Radiomic biomarkers were derived from each gross tumor volume. The biomarkers included 4 representative radiomic features from tumor first-order statistics, shape, texture, and wavelet groups, as well as a combined 4-feature signature. Univariable Cox proportional hazards models for DM risk were identified. The discriminative performance of prognostic univariable and multivariable models was compared using the concordance index (C-index). Subgroup analyses were performed. RESULTS: There were 300 HPV-related OPC patients who were eligible for the analysis. A total of 36 DM events occurred within a median follow-up period of 5 years. On univariable analysis, top results included the 4 representative radiomic features (C-index, 0.670-0.686; P < .001), the radiomic signature (C-index, 0.670; P < .001), tumor stage (C-index, 0.633; P < .001), tumor diameter (C-index, 0.653; P < .001), and tumor volume (C-index, 0.674; P < .001), which demonstrated moderate discrimination of DM risk. Combined clinical-radiomic models yielded significantly improved performance (C-index, 0.701-0.714; P < .05). In subgroup analyses, the radiomic biomarkers consistently stratified patients for DM risk, particularly for those cohorts with greater risks (C-index, 0.663-0.796), such as patients with stage III disease. CONCLUSIONS: Radiomic biomarkers appear to classify DM risk for patients with nonmetastatic HPV-related OPC. Radiomic biomarkers could be used either alone or with other clinical characteristics in the assignment of DM risk in future HPV-related OPC clinical trials.

MicroRNAs in extracellular vesicles: potential cancer biomarkers.

Extracellular vesicles (EV) are small membrane-bound structures that are secreted by various cell types, including tumor cells. Recent studies have shown that EVs are important for cell-to-cell communication, locally and distantly; horizontally transferring DNA, mRNA, microRNA (miRNA), proteins and lipids. In the context of cancer biology, tumor-derived EVs are capable of modifying the microenvironment, promoting tumor progression, immune evasion, angiogenesis and metastasis. miRNAs contained within EVs are functionally associated with cancer progression, metastasis and aggressive tumor phenotypes. These factors, along with their stability in bodily fluids, have led to extensive investigations on the potential role of circulating EV-derived miRNAs as tumor biomarkers. In this review, we summarize the current understanding of circulating EV miRNAs in human cancer, and discuss their clinical utility and challenges in functioning as biomarkers.

HPV Associated Head and Neck Cancer.

Head and neck cancers (HNCs) are a highly heterogeneous group of tumours that are associated with diverse clinical outcomes. Recent evidence has demonstrated that human papillomavirus (HPV) is involved in up to 25% of HNCs; particularly in the oropharyngeal carcinoma (OPC) subtype where it can account for up to 60% of such cases. HPVs are double-stranded DNA viruses that infect epithelial cells; numerous HPV subtypes, including 16, 18, 31, 33, and 35, drive epithelial cell transformation and tumourigenesis. HPV positive (HPV+) HNC represents a distinct molecular and clinical entity from HPV negative (HPV-) disease; the biological basis for which remains to be fully elucidated. HPV positivity is strongly correlated with a significantly superior outcome; indicating that such tumours should have a distinct management approach. This review focuses on the recent scientific and clinical investigation of HPV+ HNC. In particular, we discuss the importance of molecular and clinical evidence for defining the role of HPV in HNC, and the clinical impact of HPV status as a biomarker for HNC.

MicroRNAs in nasopharyngeal carcinoma.

It is becoming increasingly evident that aberrantly expressed microRNAs (miRNAs) are responsible for a number of disease processes, including cancer initiation and progression. miRNAs have been implicated as key players in numerous neoplasms, including nasopharyngeal carcinoma (NPC). Functionally, deregulation of miRNAs that act either as tumour suppressors or oncogenes results in numerous cancer-associated phenomena, including changes in proliferation, migration, and cell survival. Furthermore, miRNA expression has been associated with chemoresistant or radioresistant phenotypes; highlighting the importance of miRNAs in mediating oncogenic processes. Prognostic and predictive miRNA signatures have been defined for a variety of cancer types, including NPC, whereby these signatures offer a potentially important clinical tool for assessing the disease state, as well as predicting treatment response and clinical outcome. Therefore, further examination and validation of miRNAs that are deregulated in NPC will provide insight into the fundamental drivers of this disease, which will aid in the identification of novel targeted treatments. This review summarizes recent advances in the study of miRNAs in NPC, with specific discussion on the role of miRNAs in NPC pathogenesis and the potential utility of miRNAs as prognostic biomarkers. Our increasing understanding of the role of miRNAs in NPC tumorigenesis and their application as novel biomarkers will undoubtedly prove useful in the stratification of future patients into clinically relevant treatment classifications, thereby improving and personalizing disease management.

MiR-449a promotes breast cancer progression by targeting CRIP2.

The identification of prognostic biomarkers and their underlying mechanisms of action remain of great interest in breast cancer biology. Using global miRNA profiling of 71 lymph node-negative invasive ductal breast cancers and 5 normal mammary epithelial tissues, we identified miR-449a to be highly overexpressed in the malignant breast tissue. Its expression was significantly associated with increased incidence of patient relapse, decreased overall survival, and decreased disease-free survival. In vitro, miR-449a promoted breast cancer cell proliferation, clonogenic survival, migration, and invasion. By utilizing a tri-modal in silico approach for target identification, Cysteine-Rich Protein 2 (CRIP2; a transcription factor) was identified as a direct target of miR-449a, corroborated using qRT-PCR, Western blot, and luciferase reporter assays. MDA-MB-231 cells stably transfected with CRIP2 demonstrated a significant reduction in cell viability, migration, and invasion, as well as decreased tumor growth and angiogenesis in mouse xenograft models. Our data revealed that overexpression of miR-449a suppresses CRIP2, which then affects the tumor vasculature, likely via NF-κB/p65 complex-mediated transcription of VEGF. These finding define an oncogenic function of miR-449a in human breast cancer, and highlight the importance of this pathway in driving aggressive behaviour.

The complexity of microRNAs in human cancer.

MicroRNAs (miRNAs) are small non-coding RNA molecules that have key regulatory roles in cancer, acting as both oncogenes and tumor suppressors. Due to the potential roles of miRNAs in improving cancer prognostic, predictive, diagnostic and therapeutic approaches, they have become an area of intense research focus in recent years. MiRNAs harbor attractive features allowing for translation to the clinical world, such as relatively simple extraction methods, resistance to molecular degradation, and ability to be quantified. Numerous prognostic, predictive and diagnostic miRNA signatures have been developed. To date however, miRNA analysis has not been adopted for routine clinical use. The objectives of this article are to provide an overview of miRNA research and review a selection of miRNA studies in breast cancer, cervical cancer, sarcoma, and nasopharyngeal carcinoma to highlight advances and challenges in miRNA cancer research.

The microRNA-218~Survivin axis regulates migration, invasion, and lymph node metastasis in cervical cancer.

Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.

Pre-clinical characterization of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, combined with ionizing radiation for head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is over-expressed in nearly all cases of squamous cell carcinoma of the head and neck (SCCHN), and is an important driver of disease progression. EGFR targeted therapies have demonstrated clinical benefit for SCCHN treatment. In this report, we investigated the pre-clinical efficacy of Dacomitinib (PF-00299804), an irreversible pan-ErbB inhibitor, both alone and in combination with ionizing radiation (IR), a primary curative modality for SCCHN. One normal oral epithelial (NOE) and three SCCHN (FaDu, UT-SCC-8, UT-SCC-42a) cell lines were used to conduct cell viability, clonogenic survival, cell cycle, and immunoblotting assays in vitro, using increasing doses of Dacomitinib (10-500 nM), both with and without IR (2-4 Gy). The FaDu xenograft model was utilized for tumor growth delay assays in vivo, and immunohistochemical analyses were conducted on extracted tumors. A dose-dependent reduction in cell viability and clonogenic survival after Dacomitinib treatment was observed in all three SCCHN models. Treatment led to a significant reduction in EGFR signalling, with a subsequent decrease in phosphorylation of downstream targets such as ERK, AKT, and mTOR. In vivo, Dacomitinib treatment delayed tumor growth, while decreasing phospho-EGFR and Ki-67 immunoexpression. These effects were further enhanced when combined with IR, both in vitro and in vivo. The preclinical data support the further evaluations of Dacomitinib combined with IR for the future management of patients with SCCHN.

A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (UROD) catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16), was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM), but did not affect porphobilinogen deaminase (at 62.5 µM), thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE) cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1). This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

Dissecting the in vivo leukemogenic potency of BCLxl.

Overexpression of anti-apoptotic members of the BCL2 family has been found in all types of cancer. A member of the family, BCLxl (B-cell lymphoma extra-large), is known to be associated with the progression of leukemogenesis. In the present study, we focused on understanding the domains of BCLxl responsible for in vivo oncogenic potency. To this end, we utilized engineered BCLxl proteins with alternative transmembrane domains (TM) or chimeric BCLxl proteins containing domains from a less potent BCL2-like protein, BCLb. As expected, mice receiving MYC-only expressing bone marrow develop leukemia by 100 days, whereas co-expression of MYC with wild-type BCLxl led to aggressive myeloid leukemia with an average latency of ~25 days. Interestingly, mice injected with bone marrow co-expressing MYC and BCLxl targeted specifically to either mitochondria or ER also succumbed to leukemia with an average latency of ~25 days. Further, our study was extended to examine the role of the BH4 domain in driving potent leukemogenesis. Mice injected with bone marrow co-expressing MYC and BCLb succumb to leukemia in an average of ~55 days, but interestingly a BCLxl protein containing only the loop region of BCLb drove MYC-induced leukemogenesis with the same latency as wild-type BCLxl. These data suggest that the localization of exogenous BCLxl to either mitochondria or ER is not a steadfast dictator of in vivo oncogenic potency. Further, our findings suggest that the loop domain of BCLb and BCLxl is not responsible for dictating the in vivo leukemogeneic potency. This study provides further mechanistic details into the biochemical functions of BCLxl.