Citations

Abstract Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 “resistance” genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862–70. ©2017 AACR.

AbstractThe purpose of this study is to identify regions of spinal column in which more than three contiguous vertebrae can be reliably and quickly aligned within 1 mm using a 6‐degree (6D) couch and full body immobilization device. We analyzed 45 cases treated over a 3‐month period. Each case was aligned using ExacTrac x‐ray positioning system with integrated 6D couch to be within 1° and 1 mm in all six dimensions. Cone‐Beam computed tomography (CBCT) with at least 17.5 cm field of view (FOV) in the superior–inferior direction was taken immediately after ExacTrac positioning. It was used to examine the residual error of five to nine contiguous vertebrae visible in the FOV. The residual error of each vertebra was determined by expanding/contracting the vertebrae contour with a margin in millimeter integrals on the planning CT such that the new contours would enclose the corresponding vertebrae contour on CBCT. Submillimeter initial setup accuracy was consistently achieved in 98% (40/41) cases for a span of five or more vertebrae starting from T2 vertebra and extending caudally to S5. The curvature of spinal column along the cervical region and cervicothoracic junction was not easily reproducible between treatment and simulation. Fifty‐seven percent (8/14) of cases in this region had residual setup error of more than 1 mm in nearby vertebrae after alignment using 6D couch with image guidance. In conclusion, 6D couch integrated with image guidance is convenient and accurately corrects small rotational shifts. Consequently, more than three contiguous vertebrae can be aligned within 1 mm with immobilization that reliably reproduces the curvature of the thoracic and lumbar spinal column. Ability of accurate setup is becoming less a concern in limiting the use of stereotactic radiosurgery or stereotactic body radiation therapy to treat multilevel spinal target.

Abstract Background: In vivo synergy with concurrent PI3-Kinase inhibition and PARP inhibition has been observed in BRCA-deficient and BRCA-proficient preclinical models of triple negative breast cancer (TNBC) and ovarian cancer (OC). A phase I trial of the oral pan-class I PI3-Kinase inhibitor BKM120 and the PARP inhibitor olaparib demonstrated anti-cancer activity in TNBC and OC, both in patients with and without germline BRCA1 and BRCA2 (BRCA) mutations. However, CNS toxicity (depression) and liver function test abnormalities limited dose escalation of BKM120 prompting evaluation of the alpha specific PI3-Kinase inhibitor BYL719 (which has no CNS toxicity) in combination with olaparib. Methods: Olaparib was administered twice daily (tablet formulation) and BYL719 daily on a 28-day cycle, both orally. A 3 + 3 dose-escalation design was employed with primary objectives of defining the maximum tolerated dose (MTD) and recommended phase 2 dose of the combination of BYL719 and olaparib, and secondary objectives of defining toxicity, activity, and pharmacokinetic profiles of both agents. Eligibility included recurrent TNBC or high grade serous (HGS) OC, or any histology OC or breast cancer (BC) with presence of a known germline BRCA mutation, performance status of 0-1 and measurable/evaluable cancer. Patients with platinum sensitive or resistant or refractory OC were eligible and prior PARP inhibitor use was allowed. Dose-expansion cohorts at the MTD were enrolled for both BC and OC. Results: 46 patients (16 BC and 30 OC) have been enrolled in the study; 28 patients participated in the dose escalation portion of the study (4 BC and 24 OC). Two patients with OC did not receive study drugs because of ineligibility. MTD was defined as BYL719 200mg once daily and olaparib 200mg twice daily. Dose limiting toxicities included hyperglycemia, rash and fever with decreased neutrophil count. Four patients (3 OC and 1 BC) discontinued protocol therapy because of toxicity (2 for hyperglycemia, 1 for nausea and 1 for allergic reaction). Most common toxicities included nausea, hyperglycemia, fatigue, diarrhea and vomiting. At the MTD, 6 patients with OC and 12 patients with BC were enrolled into a dose expansion cohort. The OC expansion cohort has completed enrollment, while the BC cohort is still enrolling. Among patients with OC who received study drugs (28 patients, 26 (93%) with platinum resistant disease), objective response rate (ORR) by RECIST 1.1 was 36% (10/28 patients, all partial responses (PRs)). Median duration of response was 167 days (range 16-398 days); 5 of 10 patients with PR remain on treatment. ORR was 33% for patients with germline BRCA mutations and 31% for patients without germline BRCA mutations. Among patients without germline BRCA mutations with platinum resistant OC, ORR was 29%. Conclusions: Combined BYL719 and olaparib is feasible, and similar clinical benefit was observed in patients with and without germline BRCA mutations. The activity of this combination in OC patients without germline BRCA mutations and with platinum resistant disease was higher than expected from olaparib monotherapy and warrants further investigation. This work was funded in part by the Stand Up To Cancer Ovarian Dream Team. Clinical trial: NCT01623349. Citation Format: Panagiotis A. Konstantinopoulos, William T. Barry, Michael Birrer, Shannon N. Westin, Sarah Farooq, Karen Cadoo, Christin Whalen, Weixiu Luo, Hui Liu, Carol Aghajanian, David B. Solit, Gordon B. Mills, Barry S. Taylor, Helen Won, Michael F. Berger, Sangeetha Palakurthi, Joyce F. Liu, Lew Cantley, Scott H. Kaufmann, Elizabeth M. Swisher, Alan D. D'Andrea, Eric Winer, Gerburg M. Wulf, Ursula A. Matulonis. Phase I study of the alpha specific PI3-Kinase inhibitor BYL719 and the poly (ADP-Ribose) polymerase (PARP) inhibitor olaparib in recurrent ovarian and breast cancer: Analysis of the dose escalation and ovarian cancer expansion cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT008. doi:10.1158/1538-7445.AM2017-CT008

Abstract Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 +/− mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with ( S )-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

Abstract FAM46C is one of the most recurrently mutated genes in multiple myeloma; however its role in disease pathogenesis has not been determined. Here we demonstrate that wild-type (WT) FAM46C overexpression induces substantial cytotoxicity in multiple myeloma cells. In contrast, FAM46C mutations found in multiple myeloma patients abrogate this cytotoxicity, indicating a survival advantage conferred by the FAM46C mutant phenotype. WT FAM46C overexpression downregulated IRF4, CEBPB, and MYC and upregulated immunoglobulin (Ig) light chain and HSPA5/BIP. Furthermore, pathway analysis suggests that enforced FAM46C expression activated the unfolded protein response pathway and induced mitochondrial dysfunction. CRISPR-mediated depletion of endogenous FAM46C enhanced multiple myeloma cell growth, decreased Ig light chain and HSPA5/BIP expression, activated ERK and antiapoptotic signaling, and conferred relative resistance to dexamethasone and lenalidomide treatments. Genes altered in FAM46C-depleted cells were enriched for signaling pathways regulating estrogen, glucocorticoid, B-cell receptor signaling, and ATM signaling. Together these results implicate FAM46C in myeloma cell growth and survival and identify FAM46C mutation as a contributor to myeloma pathogenesis and disease progression via perturbation in plasma cell differentiation and endoplasmic reticulum homeostasis. Cancer Res; 77(16); 4317–27. ©2017 AACR.

Significance Neuromyelitis optica (NMO) is a severe inflammatory central nervous system demyelinating disease (IDD). Detection of NMO-specific IgG in serum distinguishes NMO from other IDDs, enables rapid diagnosis, and expedites NMO-appropriate therapy. Binding of NMO-IgG to aquaporin-4 (AQP4), the astrocytic water channel antigen, initiates endocytosis of AQP4 and its coupled EAAT2 glutamate transporter. Sequelae potentially contributing to NMO lesions include reduced water fluxes, reduced extracellular glutamate clearance, activation of a proinflammatory astrocyte response, and complement activation. Our paper investigates early events following IgG–AQP4 interaction and demonstrates that AQP4 and EAAT2 internalization, but not astrocytic secretion of IL-6, requires astrocytic Fc receptor signaling. Selective attenuation of these signaling events offers an upstream therapeutic target.

Abstract Purpose: Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor–positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the antiproliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies. Experimental Design: Eligible patients with clinical stage II/III ER+/HER2− breast cancer received anastrozole 1 mg daily for 4 weeks (cycle 0; with goserelin if premenopausal), followed by adding palbociclib (125 mg daily on days 1–21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67 > 10%, in which case patients went off study due to inadequate response. Anastrozole was continued until surgery, which occurred 3 to 5 weeks after palbociclib exposure. Later patients received additional 10 to 12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression, and mutation profiles. The primary endpoint was complete cell cycle arrest (CCCA: central Ki67 ≤ 2.7%). Results: Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs. C1D1 26%, P < 0.001). Palbociclib enhanced cell-cycle control over anastrozole monotherapy regardless of luminal subtype (A vs. B) and PIK3CA status with activity observed across a broad range of clinicopathologic and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with nonluminal subtypes and persistent E2F-target gene expression. Conclusions: Palbociclib is an active antiproliferative agent for early-stage breast cancer resistant to anastrozole; however, prolonged administration may be necessary to maintain its effect. Clin Cancer Res; 23(15); 4055–65. ©2017 AACR.

Purpose To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level was > 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI). Methods The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled postmenopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 determination after 2 to 4 weeks of AI. If the Ki67 was > 10%, patients were switched to neoadjuvant chemotherapy. A pCR rate of > 20% was the predefined efficacy threshold. In patients who completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence differed by PEPI = 0 score (T1 or T2, N0, Ki67 < 2.7%, ER Allred > 2) versus PEPI > 0 disease. Results Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%) of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI > 0 (recurrence hazard ratio [PEPI = 0 v PEPI > 0], 0.27; P = .014; 95% CI, 0.092 to 0.764). Conclusion Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant proliferation. The optimal therapy for these patients should be further investigated. For patients with PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A Phase III Study; clinical trial information: NCT01953588).