Pavan K. Bendapudi

Combined Inactivation of MYC and K-Ras Oncogenes Reverses Tumorigenesis in Lung Adenocarcinomas and Lymphomas

Background Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as “oncogene-addiction.” However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment. Methodology/Principal Findings To examine how the MYC and K-rasG12D oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-rasG12D to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-rasG12D- or MYC/K-rasG12D-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-rasG12D-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-rasG12D resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-rasG12D in maintenance of lung tumors, we found that the down-stream mediators of K-rasG12D signaling, Stat3 and Stat5, are dephosphorylated following conditional K-rasG12D but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-rasG12D. Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation. Conclusions/Significance Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.

Survival and Death Signals Can Predict Tumor Response to Therapy After Oncogene Inactivation

Modeling survival and death signaling in tumors by combining quantitative imaging and in situ biomarker analysis can be used to predict responses to therapy after oncogene inactivation. Predicting Oncogene Addiction with Math Some cancer cells are dependent or “addicted” to the continued activity of oncoproteins such as Myc. Drugs that target these oncoproteins induce the addicted cancer cells to die rapidly, a phenomenon called “oncogene addiction.” Marked clinical responses have been reported in some cancer patients, particularly those with lung cancer, after treatment with drugs targeting oncoproteins. However, only a distinct subset of human cancer patients have tumors that exhibit this behavior of oncogene addiction. The ability to predict when a tumor will exhibit oncogene addiction would be useful not only for developing new oncoprotein-targeted therapies but also for selecting which cancer patients are likely to respond best to such drugs. In a new study, Tran et al. use a systems approach to mathematically model the behavior of oncogene-addicted lung and lymphoma tumors. Starting from the simple premise that oncogene addiction could be deduced from the balance between converging signals from cell survival and cell death pathways in the tumors, Tran and colleagues were able to predict the clinical behavior of lung and lymphoma cells in mice treated with oncoprotein-targeted therapies. They also showed that their mathematical model for oncogene addiction could predict the distinct temporal relationships of signaling molecules from converging survival and death pathways. The group then used quantitative imaging of mouse tumors to train an algorithm called support vector machine to accurately differentiate mice that responded to oncoprotein-targeted therapy from those mice that did not respond. They then extended this quantitative imaging approach to humans with lung cancer who had been treated with oncoprotein-targeted therapy. Their model was able to differentiate patients who had lung tumors that were oncogene-addicted and hence were most likely to benefit from this treatment from those who did not. Although much more work needs to be done, the Tran et al. study demonstrates that mathematical modeling could be a useful tool for helping guide the management of cancer patients. Qualitative approaches alone are not sufficient given the complexity and heterogeneity of tumors, so the ability to quantify complex systems will be of benefit to the field of oncology. Finally, quantitative imaging combined with classifier algorithms may be useful clinically for distinguishing those cancer patients who are most likely to derive clinical benefit from oncoprotein-targeted therapeutics. Cancers can exhibit marked tumor regression after oncogene inhibition through a phenomenon called “oncogene addiction.” The ability to predict when a tumor will exhibit oncogene addiction would be useful in the development of targeted therapeutics. Oncogene addiction is likely the consequence of many cellular programs. However, we reasoned that many of these inputs may converge on aggregate survival and death signals. To test this, we examined conditional transgenic models of K-rasG12D– or MYC-induced lung tumors and lymphoma combined with quantitative imaging and an in situ analysis of biomarkers of proliferation and apoptotic signaling. We then used computational modeling based on ordinary differential equations (ODEs) to show that oncogene addiction could be modeled as differential changes in survival and death intracellular signals. Our mathematical model could be generalized to different imaging methods (computed tomography and bioluminescence imaging), different oncogenes (K-rasG12D and MYC), and several tumor types (lung and lymphoma). Our ODE model could predict the differential dynamics of several putative prosurvival and prodeath signaling factors [phosphorylated extracellular signal–regulated kinase 1 and 2, Akt1, Stat3/5 (signal transducer and activator of transcription 3/5), and p38] that contribute to the aggregate survival and death signals after oncogene inactivation. Furthermore, we could predict the influence of specific genetic lesions (p53−/−, Stat3-d358L, and myr-Akt1) on tumor regression after oncogene inactivation. Then, using machine learning based on support vector machine, we applied quantitative imaging methods to human patients to predict both their EGFR genotype and their progression-free survival after treatment with the targeted therapeutic erlotinib. Hence, the consequences of oncogene inactivation can be accurately modeled on the basis of a relatively small number of parameters that may predict when targeted therapeutics will elicit oncogene addiction after oncogene inactivation and hence tumor regression.

A substrate-driven allosteric switch that enhances PDI catalytic activity

Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a–b–b′–x–a′, wherein the thioredoxin-like a and a′ domains mediate disulfide bond shuffling and b and b′ domains are substrate binding. The b′ and a′ domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b′. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a′ by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains.

Getting at MYC through RAS

The discovery of oncogenes provided insight into the molecular underpinnings of cancer and suggested the promise of novel molecular strategies for cancer treatment as highlighted in this issue by Yaari et al. and by Bishop previously ([1][1], [2][2]). However, only recently have effective drugs that

Intraoperative radiation therapy for locally advanced and recurrent soft-tissue sarcomas in adults.

PURPOSE To analyze the outcomes of and identify prognostic factors for patients treated with surgery and intraoperative radiotherapy (IORT) for locally advanced and recurrent soft-tissue sarcoma in adults from a single institution. METHODS AND MATERIALS We retrospectively reviewed 50 consecutive patients treated with IORT to 62 sites of disease. Primary sites included retroperitoneum-pelvis (78%), extremity (8%), and other (14%). Seventy percent of patients had recurrent disease failing prior surgery (70%) and/or radiation (32%). Mean disease-free interval (DFI) before IORT was 1.9 years (range, 2 weeks-5.4 years). The IORT was delivered with orthovoltage X-rays using individually sized beveled cone applicators. Clinical characteristics were as follows: mean tumor size, 10 cm (range, 1-25 cm); high-grade histologic subtype (72%); and mean dose, 1,159 cGy (range, 600-1,600 cGy). Postoperative radiation or chemotherapy was administered to 37% of IORT Sites and 32% of patients, respectively. Outcomes measured were infield control (IFC), locoregional control (LRC), distant metastasis-free survival (DMFS), disease-specific survival (DSS), and treatment-related complications. Mean and median follow-up of alive patients were 59 and 35 months, respectively. RESULTS Kaplan-Meier 5-year IFC, LRC, DMFS, and DSS probabilities for the entire group were 55%, 26%, 51%, and 25%, respectively. Prognostic factors found to be significant (p < 0.05) on multivariate analysis were prior DFI and tumor size for LRC, extremity location and leiomyosarcoma histologic subtype for DMFS, and prior DFI for DSS. Our cohort had five Grade 3/4 complications associated with treatment or a 5-year Kaplan-Meier Grade 3/4 complication-free survival rate of 85%. CONCLUSIONS IORT after tumor reductive surgery is well tolerated and seems to confer IFC in carefully selected patients.

Derivation and external validation of the PLASMIC score for rapid assessment of adults with thrombotic microangiopathies: a cohort study

BACKGROUND Among the syndromes characterised by thrombotic microangiopathy, thrombotic thrombocytopenic purpura is distinguished by a severe deficiency in the ADAMTS13 enzyme. Patients with this disorder need urgent treatment with plasma exchange. Because ADAMTS13 activity testing typically requires prolonged turnaround times and might be unavailable in resource-poor settings, a method to rapidly assess the likelihood of severe ADAMTS13 deficiency is needed. METHODS All consecutive adult patients presenting to three large academic medical centres in Boston, MA, USA, with thrombotic microangiopathy and a possible diagnosis of thrombotic thrombocytopenic purpura between Jan 8, 2004, and Dec 6, 2015, were included in an ongoing multi-institutional registry (the Harvard TMA Research Collaborative). Univariate analysis was used to identify covariates for a logistic regression model predictive of severe ADAMTS13 deficiency (≤10% activity). A clinical point score was generated, and its diagnostic performance was assessed using internal and external validation cohorts and compared to clinical assessment alone. FINDINGS 214 patients with thrombotic microangiopathy were included in the derivation cohort. A seven-component clinical prediction tool, termed the PLASMIC score, was developed and found to reliably assess the pretest probability of severe ADAMTS13 deficiency (C statistic 0·96, 95% CI 0·92-0·98). Our diagnostic model was reproducibly accurate in both the internal (0·95, 0·91-0·98) and external (0·91, 0·85-0·95) validation cohorts. The scoring system also more consistently diagnosed thrombotic microangiopathy due to severe ADAMTS13 deficiency than did standard clinical assessment, as measured by C statistic (0·96, 95% CI 0·92-0·98 for PLASMIC vs 0·83, 0·77-0·88 for clinical assessment; p<0·0001) and mean Brier score (0·065 for PLASMIC vs 0·111 for clinical assessment; mean paired difference 0·05, 95% CI 0·01-0·08; p<0·0001). When utilised in addition to clinical assessment, the PLASMIC score contributed significant discriminatory power (integrated discrimination improvement 0·24, 95% CI 0·11-0·37). INTERPRETATION We have developed and validated a clinical prediction tool-the PLASMIC score-to stratify patients with thrombotic microangiopathy according to their risk of having severe ADAMTS13 deficiency. We have shown that this scoring system is superior to standard clinical assessment in addressing the diagnostic challenge presented by thrombotic microangiopathy. Its use, together with clinical judgment, may facilitate treatment decisions in patients for whom timely results of ADAMTS13 activity testing are unavailable. FUNDING The Luick Family Fund of Massachusetts General Hospital.

Impact of severe ADAMTS13 deficiency on clinical presentation and outcomes in patients with thrombotic microangiopathies: the experience of the Harvard TMA Research Collaborative

The Harvard TMA Research Collaborative is a multi‐institutional registry‐based effort to study thrombotic microangiopathies (TMA). Laboratory and clinical parameters were recorded for 254 cases of suspected autoimmune thrombotic thrombocytopenic purpura (TTP). Patients with severe ADAMTS13 deficiency (activity ≤10%, N = 68) were more likely to be young, female and without a history of cancer treatment or transplantation. While all patients with severe deficiency were diagnosed with autoimmune TTP, those without severe deficiency frequently had disseminated intravascular coagulation, drug‐associated TMA and transplant‐related TMA. Patients with severe ADAMTS13 deficiency had superior overall survival at 360 d compared to those without severe deficiency (93·0% vs. 47·5%, P < 0·0001). Almost all patients with severe deficiency received therapeutic plasma exchange (TPE), but the use of TPE in patients with ADAMTS13 activity >10% varied significantly across the institutions in our consortium (13·2–63·8%, P < 0·0001). Nevertheless, 90‐d mortality was not different in patients with ADAMTS13 activity >10% between the three hospitals (P = 0·98). Our data show that patients with severe ADAMTS13 deficiency represent a clinically distinct cohort that responds well to TPE. In contrast, TMA without severe ADAMTS13 deficiency is associated with increased mortality that may not be influenced by TPE.

Treatment with or without plasma exchange for patients with acquired thrombotic microangiopathy not associated with severe ADAMTS13 deficiency: a propensity score–matched study

Therapeutic plasma exchange (TPE) is a proven treatment for thrombotic thrombocytopenic purpura (TTP) characterized by severe ADAMTS13 deficiency, but the efficacy of TPE in suspected TTP with an ADAMTS13 activity level of more than 10% remains controversial.

18F and 18FDG PET imaging of osteosarcoma to non-invasively monitor in situ changes in cellular proliferation and bone differentiation upon MYC inactivation

Osteosarcoma is one of the most common pediatric cancers. Accurate imaging of osteosarcoma is important for proper clinical staging of the disease and monitoring of the tumor’s response to therapy. The MYC oncogene has been commonly implicated in the pathogenesis of human osteosarcoma. Previously, we have described a conditional transgenic mouse model of MYC-induced osteosarcoma. These tumors are highly invasive and are frequently associated with pulmonary metastases. In our model, upon MYC inactivation osteosarcomas lose their neoplastic properties, undergo proliferative arrest, and differentiate into mature bone. We reasoned that we could use our model system to develop non-invasive imaging modalities to interrogate the consequences of MYC inactivation on tumor cell biology in situ. We performed Positron Emission Tomography (PET) combining the use of both 18F-fluorodeoxyglucose (18FDG) and 18F-flouride (18F) to detect metabolic activity and bone mineralization/remodeling. We found that upon MYC inactivation, tumors exhibited a slight reduction in uptake of 18FDG and a significant increase in the uptake of 18F along with associated histological changes. Thus, these cells have apparently lost their neoplastic properties based upon both examination of their histology and biologic activity. However, these tumors continue to accumulate 18FDG at levels significantly elevated compared to normal bone. Therefore, PET can be used to distinguish normal bone cells from tumors that have undergone differentiation upon oncogene inactivation. In addition, we found that 18F is a highly sensitive tracer for detection of pulmonary metastasis. Collectively, we conclude that combined modality PET/CT imaging incorporating both 18FDG and 18F is a highly sensitive means to non-invasively measure osteosarcoma growth and the therapeutic response, as well as to detect tumor cells that have undergone differentiation upon oncogene inactivation

An Algorithmic Approach to the Diagnosis and Management of the Thrombotic Microangiopathies

No clinical consult raises the heart rate of a transfusion medicine specialist quite like that involving thrombotic microangiopathy (TMA) concerning for thrombotic thrombocytopenic purpura (TTP). The differential diagnosis is complex and challenging, the patients tend to be very ill, and effective treatment requires providing therapy directed at the underlying pathology as quickly as possible. Clinical pathologists are well positioned to provide guidance in these cases since they are usually alerted to all patients in the hospital with suspected TTP and therefore have considerable experience to offer. In contrast, the consulting physician may be somewhat less experienced in formulating a diagnostic and therapeutic plan of care for these patients and might opt for therapeutic plasma exchange (TPE) when in fact a more careful evaluation is merited and other therapies might be more effective. The article by Williams and Marques1 in this issue of the Journal highlights the key role played by the clinical pathologist in the care of patients with TMA, focusing on the important distinction between TTP, hemolytic uremic syndrome (HUS), and atypical uremic syndrome (aHUS) and providing a clear and logical approach to these disorders. TMA should be viewed as a manifestation of diverse pathologies involving either hemostasis (inappropriate platelet activation or thrombin generation) or the endothelium (exposure to Shiga toxin, uncontrolled complement deposition).2 Treatments differ depending on the underlying mechanism of disease. Whereas TPE is an effective treatment for TTP,3,4 eculizumab has emerged as an important new therapy for aHUS,5 providing superior renal survival in comparison to outcomes observed following treatment with TPE alone.6 The understanding that aHUS results from dysregulation of the alternative complement pathway and is associated with inherited mutations in complement regulatory proteins while TTP results from severe deficiency of the ADAMT13 enzyme caused by autoantibodies to this … Corresponding author: Robert S. Makar, MD, PhD, Massachusetts General Hospital Blood Transfusion Service, GRJ-148, 55 Fruit St, Boston, MA 02114; rmakar{at}mgh.harvard.edu.