Significant Prolongation Of Survival Research Articles

Abstract PR-007: Targeting FASN improves type I interferon responses in irradiated glioblastoma

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with a median survival of 11-15 months. Radiotherapy (RT), the standard of care for GBM, increases adjuvanticity of cancer cells, by at least generating cancer cell intrinsic type I interferon (IFN-I) responses to jumpstart antitumor immunity. However, RT can also exacerbate potent immune inhibitory mechanisms to facilitate immune evasion. Among which, metabolic reprogramming of irradiated GBM represents an emerging mechanism of immune resistance. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is a hallmark of GBM that is emerging as a mechanisms of therapy resistance and immune escape. Because FASN was found to impair IFN-I in antiviral immunity, we hypothesize that FASN is preventing RT-induced IFN-I responses to promote GBM survival and evade immune recognition. To test this hypothesis, we first defined RT-induced metabolic changes by seahorse assay and metabolomics in the GL261 murine GBM model. We observed an increase in mitochondrial respiration, glycolysis and in lipid metabolism-related pathways in 10 gray (Gy) irradiated GL261 cells. Consistent with this last observation, we found upregulation of FASN expression by western blot and intracellular neutral lipid accumulation by BODIPY staining and transmission electron microscopy (TEM) in irradiated GL261 cells (10 Gy). Confirming the role of FASN, RT-induced accumulation of neutral lipids was reverted when GL261 cells were incubated with a pharmacologic FASN inhibitor. Next, to ask whether FASN was impairing IFN-I in irradiated GBM, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS). As expected, irradiation of GL261shNS cells enhanced the secretion of IFN-I cytokines. Strikingly, we found that FASN knockdown spontaneously induced IFN-beta and CXCL10 secretion, and this effect was even more pronounced when cells were irradiated. Finally, GL261shNS and GL261shFASN cells were orthotopically implanted in mice and IFN-I signaling was blocked using an antibody against the interferon alpha/beta receptor (IFNAR). FASN knockdown alone was sufficient to increase the median survival of mice bearing GL261shFASN tumors by 51 days vs. 35 days for GL261shNS tumors. Moreover, 33% of mice bearing GL261 tumors unable to express FASN remained tumor free for up to 100 days, while none of the GL261shNS-tumor bearing animal survived. This significant survival prolongation of mice bearing GL261shFASN tumor was completely abrogated when IFNAR was blocked. Overall, our findings suggest that FASN-mediated lipogenesis prevents RT-induced cancer cell intrinsic IFN-I to promote GBM survival. Consequently, it is possible that FASN acts as an immuno-metabolic checkpoint capable to regulate the immune system upon metabolic cues generated by RT. Citation Format: Mara De Martino, Camille Daviaud, Claire Vanpouille-Box. Targeting FASN improves type I interferon responses in irradiated glioblastoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-007.

DDRE-26. THE IMMUNO-METABOLIC ENZYME FASN PREVENTS CANCER-CELL INTRINSIC TYPE I INTERFERON RESPONSES IN GLIOBLASTOMA

Glioblastoma (GBM) is a devastating primary brain cancer with a median survival of 11–15 months. Radiation therapy (RT), the standard of care for GBM, can generate type I interferon responses (IFN-I) to jumpstart antitumor immunity. However, these effects are sometimes mitigated by inhibitory mechanisms that are exacerbated by RT. RT can modify GBM metabolism to promote de novo lipogenesis via the fatty acid synthase (FASN). Because FASN was found to impair IFN-I in antiviral immunity, we hypothesize that FASN is preventing RT-induced IFN-I responses to promote GBM survival and evade immune recognition. We first defined RT-induced metabolic changes in the GL261 murine GBM model. We observed an increase in mitochondrial respiration, glycolysis and in lipid metabolism-related pathways in 10 gray (Gy) irradiated GL261 cells. Additionally, we found upregulation of FASN by western blot and lipid accumulation by BODIPY staining in 10 Gy-GL261 cells. RT-induced lipid accumulation was reverted when GL261 cells were incubated with a FASN inhibitor. Next, to ask whether FASN was impairing IFN-I, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS). As expected, irradiation of GL261shNS cells enhanced the secretion of IFN-beta and CXCL10. This effect was more pronounced when FASN was abrogated in GL261 independently from the presence of RT. Finally, GL261shNS and GL261shFASN cells were orthotopically implanted in mice and IFN-I signaling was blocked by anti-IFN-I receptor antibody (a-IFNAR). Mice bearing GL261shFASN tumors presented a median survival of 51 days vs. 35 days for GL261shNS tumors, a significant prolongation of mice survival that was completely abrogated with a-IFNAR treatment. Overall, our findings suggest that FASN-mediated lipogenesis prevents RT-induced cancer cell intrinsic IFN-I to promote GBM survival. Consequently, it is possible that FASN acts as an immuno-metabolic checkpoint capable to regulate the immune system upon metabolic cues generated by RT.

Abstract PS10-16: Real-world efficacy of ribociclib + aromatase inhibitor/fulvestrant, or endocrine monotherapy, or chemotherapy as first-line treatment in women with HR-positive, HER2-negative locally advanced or metastatic breast cancer: Third interim analysis from the RIBANNA study

Abstract Background: In MONALEESA-3 and MONALEESA-7 trials, ribociclib (RIB, a selective CDK4/6 inhibitor) in combination with endocrine therapy (aromatase inhibitor [AI] or fulvestrant [FUL]) demonstrated a significant prolongation of overall survival among patients with breast cancer (BC), regardless of menopausal status and treatment-line. In the premenopausal or perimenopausal women, RIB + AI/FUL should be combined with a luteinizing hormone-releasing hormone agonist. The real-world evidence for the effectiveness, safety, and tolerability of RIB + AI/FUL in the routine clinical practice is needed to support the use of this combination. Methods: RIBANNA is a prospective, non-interventional study ongoing in Germany since October 2017. Pre-, peri- and postmenopausal women (planned, n = 3020) treated with RIB + AI/FUL, or endocrine monotherapy (ET), or chemotherapy (CT) as the first-line treatment for HR+/HER2- aBC in accordance with German guidelines were included. Data from routine clinical practice in all 3 cohorts, including further lines of sequential therapy, were collected. Updated data on baseline demographics and safety analysis will be presented. Additionally, first experience with CANKADO, an innovative digital and patient-friendly application for electrocardiogram (ECG) measurement, are described. By combining this app to Kardia mobile (AliveCor’s single lead device), single-lead ECG can be recorded and sent via the CANKADO app for a cardiologist to read over. The cardiologist reviews the measurements, and reports the corrected QT interval by Fredericia (QTcF) results towards the trial site. Impact of the use of CANKADO onto the clinical routine will be analyzed. Results: For the second interim analysis, 1141 patients were enrolled until July 12th, 2019, while the full analysis set comprised 813 patients (Table 1). Median duration of RIB exposure in patients on RIB + AI/FUL vs total population was 151 vs 150 days respectively. At cut-off date, patients received first-line (89%, n = 1016), second-line (7%, n = 82), and third-line treatments (1%, n = 11). A starting dose of 600 mg per day for RIB in the first-line setting was seen in most of the patients (85%); data of second-line patients will be reported later. The first-line mean daily dose of RIB was 451.6 mg/day RIB was prescribed mainly in combination with letrozole (75%), fulvestrant (12%), anastrozole (7%), and exemestane (6%). A total of 62% of patients in the study were documented as therapy-naïve for ET. Of 424 patients with early breast cancer, 129 (30%) had documented prior CT without ET. The most common all-grade adverse events frequently noted in RIB + AI/FUL cohort in comparison with other cohorts were nausea (RIB [19%] vs ET [9%] vs CT [11%]), neutropenia (19% vs 1% vs 10%), fatigue (17% vs 9% vs 14%), and leukopenia (14% vs 4% vs 12%). The usage of CANKADO application to assess QTcF was initiated in the year 2019 and first data will be presented. Conclusion: RIBANNA study showed diverse population characteristics among the patients who received RIB treatment in a real-world setting. The third interim analysis is planned in October 2020, and the updated baseline data, information on safety, digital ECG measurement using CANKADO application will be presented. Baseline Demographic Characteristics as of Second Interim AnalysisDemographic VariableRIB + AI/FUL (n = 658)Endocrine Therapy (n = 78)Chemotherapy (n = 77)Mean age, years (SD)67 (11)72 (12)62 (10)Mean time since initial diagnosis, years6.17.23.6T stage at baseline*, n (%)TX204 (31)28 (36)18 (23)T0+T1132 (20)12 (15)11 (14)T2-T4302 (46)35 (45)42 (54)N stage at baseline*, n (%)NX212 (32)29 (37)28 (36)N0+N1317 (48)36 (46)35 (46)N2+N3108 (16)10 (13)8 (10)M stage at baseline*, n (%)MX6 (1)0 (0)0 (0)M018 (3)1 (1)1 (1)M1616 (94)75 (96)71 (92)Metastatic location at baseline*, n (%)CNS, liver, lungs258 (39)14 (18)42 (54)Bone only181 (28)42 (54)11 (14)Skin, lymph nodes, other149 (23)16 (20)14 (18)*Remaining cases are missing.CNS, central nervous system. Citation Format: Achim Wöckel, Cosima Brucker, Thomas Decker, Jörg Falbrede, Helmut Forstbauer, Thomas Göhler, Oliver Hoffmann, Christian Jackisch, Jan Janssen, Andreas Köhler, Kerstin Lüdtke-Heckenkamp, Diana Lüftner, Frederik Marmé, Arnd Nusch, Toralf Reimer, Christian Roos, Marcus Schmidt, Rudolf Weide, Peter Fasching. Real-world efficacy of ribociclib + aromatase inhibitor/fulvestrant, or endocrine monotherapy, or chemotherapy as first-line treatment in women with HR-positive, HER2-negative locally advanced or metastatic breast cancer: Third interim analysis from the RIBANNA study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-16.

Results of biomarker analysis from phase II trial of nivolumab in refractory biliary tract cancer.

338 Background: Our previous phase II study demonstrated that nivolumab provides modest but durable clinical efficacy in patients (pts) with refractory biliary tract cancer (BTC), suggesting the significant clinical benefit of nivolumab in selected pts and identification of potential predictive biomarkers. Here, we report outcomes from the biomarker analysis. Methods: Pre-treatment tumor samples were obtained and assessed for immunohistochemistry (IHC) with PD-1 and PDL-1 antibodies, mRNA sequencing (RNAseq), and whole exome sequencing (WES). Clinical efficacy was correlated with the molecular analysis. Prior cisplatin exposure was reviewed to evaluate the impact of cisplatin on the tumor mutational burden (TMB) and clinical outcome. Results: Among 46 pts who had tumor response evaluation, 31 pts received cisplatin prior to nivolumab, and 15 were cisplatin-naive. Pre-treatment tumor samples were assessed for IHC (n = 42), RNAseq (n = 11), and WES (n = 11) based on tissue availability. There was no statistically significant correlation between prior cisplatin exposure and clinical outcome. Among 42 available tumor samples, 18 (43%) expressed PD-L1 positivity ( > 1%), which was associated with a statistically significant prolonged progression free survival (PFS). Median PFS was 10.4 months for PD-L1 positive vs 2.4 months for PD-L1 negative (HR 0.23, 95% CI 0.10-0.51; P < 0.001), while there was no statistically significant correlation between PD-1 expressing tumor infiltrating lymphocytes and clinical outcome. There was also no statistically significant difference in TMB of cisplatin-exposed (n = 8) vs. cisplatin-naïve tumor samples (n = 3) (9162, SD = 150 vs. 8898, SD = 806, p = 0.62). In comparing the group with prolonged disease control of at least 16 weeks (n = 4) to those with rapid disease progression (n = 7) who had mRNA sequencing performed, levels of AC005609.1, FAT3, TMEM151A, ADARB2, FAM153A were all significantly upregulated (p < 0.01), while levels of CLCA1, MUC2, IGHV3-43, SWORA6, and CRISP3 were all significantly downregulated (p < 0.01). Conclusions: Prior cisplatin exposure did not lead to statistically significant differences in clinical outcome or TMB in advanced BTC pts treated with nivolumab. However, PD-L1 expression > 1% correlated with improved PFS, and variations in level of certain mRNA sequences were noted when comparing pts who had rapid disease progression vs. prolonged disease control with nivolumab. This suggests that BTC pts with positive PD-L1 expression and a particular mRNA profile may have a favorable response to nivolumab. Further studies are needed to confirm these findings. Clinical trial information: NCT02829918.

Induction Chemotherapy for Primarily Unresectable Locally Advanced Pancreatic Adenocarcinoma-Who Will Benefit from a Secondary Resection?

Background and Objectives: An increasing number of patients (pts) with locally advanced pancreatic cancer (LAPC) are treated with an intensive neoadjuvant therapy to obtain a secondary curative resection. Only a certain number of patients benefit from this intention. The aim of this investigation was to identify prognostic factors which may predict a benefit for secondary resection. Materials and Methods: Survival time and clinicopathological data of pts with pancreatic cancer were prospective and consecutively collected in our Comprehensive Cancer Center Database. For this investigation, we screened for pts with primarily unresectable pancreatic cancer who underwent a secondary resection after receiving induction therapy in the time between March 2017 and May 2019. Results: 40 pts had a sufficient database to carry out a reliable analysis. The carbohydrate-antigen 19-9 (CA 19-9) level of the pts treated with induction therapy decreased by 44.7% from 4358.3 U/mL to 138.5 U/mL (p = 0.001). The local cancer extension was significantly reduced (p < 0.001), and the Eastern Cooperative Oncology Group (ECOG) performance status was lowered (p = 0.03). The median overall survival (mOS) was 20 months (95% CI: 17.2–22.9). Pts who showed a normal CA 19-9 level (<37 U/mL) at diagnosis and after neoadjuvant therapy or had a Body Mass Index (BMI) below 25 kg/m2 after chemotherapy had a significant prolonged overall survival (29 vs. 19 months, p = 0.02; 26 vs. 18 months, p = 0.04; 15 vs. 24 months, p = 0.01). Pts who still presented elevated CA 19-9 levels >400 U/mL after induction therapy did not profit from a secondary resection (24 vs. 7 months, p < 0.001). Nodal negativity as well as the performance of an adjuvant therapy lead to better mOS (25 vs. 15 months, p = 0.003; 10 vs. 25 months, p < 0.001). Conclusion: The pts in our investigation had different benefits from the multimodal treatment. We identified the CA 19-9 level at time of diagnosis and after neoadjuvant therapy as well as the preoperative BMI as predictive factors for overall survival. Furthermore, diagnostics of presurgical nodal status should gain more importance as nodal negativity is associated with better outcome.

Local hyperthermia combined with CTLA-4 blockade induces both local and abscopal effects in a murine breast cancer model

Purpose To evaluate the antitumor efficacy in local and distant tumors induced by local hyperthermia with CTLA-4 blockade Methods A mouse breast cancer cell line was inoculated into both sides of the legs of mice. The mice were treated with three administrations of CTLA-4 blockade, a single application of local hyperthermia (42.5 °C for 20 min) to the tumor on one side of the leg, or the combination of the two. Tumor growth in locally heated tumors (HT tumors) and unheated distant tumors (UnHT tumors) and overall survival were evaluated. Results In the combination group, tumor volume significantly decreased for both HT and UnHT tumors compared with the tumors in the untreated and local hyperthermia monotherapy groups. Remarkable efficacy was only observed in the combination therapy group, in which 7 of 18 mice responded to HT and UnHT tumors, with significant prolonged overall survival. Conclusions Combination therapy enhanced the antitumor response not only in HT tumors but also in UnHT tumors and prolonged overall survival.

Endosomal Escapable Cryo-Treatment Driven Membrane Encapsulated Ga Liquid-Metal Transformer to Facilitate Intracellular Therapy

Metallic/nonmetallic shape transformable materials hold big promise for improved intracellular therapy with efficient endosomal escape in tumor treatments. However, until now, there are still rather limited biomedical practices for such materials which are mainly attributed to their inherent uncontrolled morphological transformation, difficult metabolization, accumulated toxicity, non-selective destruction and difficult real-time imaging. In this study, Ga-based liquid metal, with distinct properties of high thermal conductivity, excellent phase transition, injectability, targetability, easy-preparation and biosafety, is found to exhibit remarkable transformation from sphere shape to cactus-like structure in micro-scale under freezing. Particularly, it is revealed that only cell membrane-encapsulated Ga particles (Ga/MPs) display dramatic shape variation in cooling under Cryo-TEM contrasting to pure Ga particles (GaPs). Following that, the cryo-triggered Ga/MPs transformers are designed for effective endosomal escape via physical-mechanical strategy to disrupt endosomal membrane which lead to highly efficient cancer cell killing. Moreover, under in vivo antitumor treatments settings, Ga/MPs exhibit significant tumor growth inhibition and prolonged survival time to evaluate the collaborative efficacy of cryoablation and endosomal escape mechanism, as well high-resolution in vivo CT imaging is achieved. This study opens a favorable and versatile strategy based on Ga particle transformer to assist high-performance precise intracellular therapy in future tumor therapeutics.

225Ac-labeled CD33-targeting antibody reverses resistance to Bcl-2 inhibitor venetoclax in acute myeloid leukemia models.

ABSTRACTPurposeDespite the availability of new drugs, many patients with acute myeloid leukemia (AML) do not achieve remission and outcomes remain poor. Venetoclax is a promising new therapy approved for use in combination with a hypomethylating agent or with low‐dose cytarabine for the treatment of newly diagnosed older AML patients or those ineligible for intensive chemotherapy. 225Actinium‐lintuzumab (225Ac‐lintuzumab) is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single‐agent activity in relapsed/refractory AML. Increased expression of MCL‐1 is a mediator of resistance to venetoclax in cancer.Experimental designHere we investigated the potential for 225Ac‐lintuzumab‐directed DNA damage to suppress MCL‐1 levels as a possible mechanism of reversing resistance to venetoclax in two preclinical in vivo models of AML.ResultsWe demonstrated that 225Ac‐lintuzumab in combination with venetoclax induced a synergistic increase in tumor cell killing compared to treatment with either drug alone in venetoclax‐resistant AML cell lines through both an induction of double‐stranded DNA breaks (DSBs) and depletion of MCL‐1 protein levels. Further, this combination led to significant tumor growth control and prolonged survival benefit in venetoclax‐resistant in vivo AML models.ConclusionsThere results suggest that the combination of 225Ac‐lintuzumab with venetoclax is a promising therapeutic strategy for the treatment of patients with venetoclax‐resistant AML.Clinical trial of this combination therapy (NCT03867682) is currently ongoing.

The Therapeutic Effects of Dodecaborate Containing Boronophenylalanine for Boron Neutron Capture Therapy in a Rat Brain Tumor Model.

Simple SummaryWe have developed a new boron compound for application in boron neutron capture therapy (BNCT) named boronophenylalanine–amide alkyl dodecaborate (BADB). It is characterized by a larger amount of 10B per molecule, linking boronphenylalanine (BPA) and dodecaborate, and we conducted various experiments on its efficacy. Its high accumulation at the cellular level made it a promising novel drug, but it did not sufficiently accumulate in brain tumor tissue when intravenously administered. However, in neutron irradiation experiments, the drug showed remarkably high compound biological effectiveness and significantly prolonged the survival time in rat brain tumor models. We confirmed the antitumor efficacy of BADB in BNCT and its additional efficacy when administered in combination with BPA. Though this drug showed poor results when administered as a single agent, it was superior to BPA alone when administered in combination with BPA, making it a drug that we have been waiting for in our clinical practice.Background: The development of effective boron compounds is a major area of research in the study of boron neutron capture therapy (BNCT). We created a novel boron compound, boronophenylalanine–amide alkyl dodecaborate (BADB), for application in BNCT and focused on elucidating how it affected a rat brain tumor model. Methods: The boron concentration of F98 rat glioma cells following exposure to boronophenylalanine (BPA) (which is currently being utilized clinically) and BADB was evaluated, and the biodistributions in F98 glioma-bearing rats were assessed. In neutron irradiation studies, the in vitro cytotoxicity of each boron compound and the in vivo corresponding therapeutic effect were evaluated in terms of survival time. Results: The survival fractions of the groups irradiated with BPA and BADB were not significantly different. BADB administered for 6 h after the termination of convection-enhanced delivery ensured the highest boron concentration in the tumor (45.8 μg B/g). The median survival time in the BADB in combination with BPA group showed a more significant prolongation of survival than that of the BPA group. Conclusion: BADB is a novel boron compound for BNCT that triggers a prolonged survival effect in patients receiving BNCT.

TAMI-49. THE ALTERNATIVE SPLICING FACTOR MBNL1 INHIBITS GLIOBLASTOMA TUMOR INITIATION AND PROGRESSION BY REDUCING HYPOXIA-INDUCED STEMNESS

Abstract Muscleblind-like-proteins (MBNL) belong to a family of tissue-specific RNA metabolism-regulators that control pre-messenger RNA-splicing (AS). Inactivation of MBNL causes an adult-to-fetal AS transition, resulting in the development of myotonic dystrophy. We have previously shown that the aggressive brain cancer, glioblastoma (GBM), maintains stem-like features (GSC) through hypoxia-induced responses. Accordingly, we hypothesized that the hypoxia-induced responses in GBM might also include MBNL-based AS to promote tumor progression. When cultured in hypoxia, GSCs rapidly export MBNL1 out of the nucleus resulting in significant inhibition of MBNL1 activity. Notably, the hypoxia-regulated inhibition of MBNL1 also resulted in evidence of adult-to-fetal alternative splicing transitions. Forced expression of a constitutively active isoform of MBNL1 inhibited GSC self-renewal and tumor initiation in orthotopic transplantation models. Using a tetracycline-inducible system, induced expression of MBNL1 in established orthotopic tumors dramatically inhibited tumor progression resulting in a significant prolongation of survival. This study reveals that MBNL1 plays an essential role in GBM stemness and tumor progression, whereby hypoxic responses within the tumor inhibit MBNL1 activity, promoting stem-like phenotypes and tumor growth. Reversing these effects on MBNL1 may, therefore, yield potent tumor-suppressor activities, uncovering new therapeutic opportunities to counter this devastating disease.

CTNI-73. OVERALL SURVIVAL OF NEWLY DIAGNOSED GLIOBLASTOMA PATIENTS TREATED BY STANDARD THERAPY IN COMPARISON TO STANDARD THERAPY PLUS TUMOR TREATING FIELDS

Abstract BACKGROUND Tumor treating fields (TTFields; 200 kHz) have shown significant prolonged survival in newly diagnosed (ndGBM). TTFields are anti-mitotic, low intensity, intermediate frequency alternating electric fields. The applied fields disrupt the mitotic spindle, microtubule assembly and the segregation of intracellular organelles during cell division, leading to apoptosis or mitotic arrest. We compared overall survival (OS) between patients recently treated with standard therapy and standard therapy plus TTFields at our institution. METHODS Subjects (N=25) with ndGBM treated with standard therapy plus TTFields (STDTh-TTF) at our institution were included. Standard therapy (STDTh) consisted of surgical resection, followed by combined radiotherapy and chemotherapy (Temozolomide). In 3 cases, biopsies were performed instead of resection. The date of resection or biopsy was considered the entry date and was used in calculating survival. The study took place from July 2015 to April 2019. A matching control group of 25 subjects with ndGBM were treated with STDTh alone at our institution and were assembled from our database based primarily on date of resection or biopsy, and secondarily by age (2 subjects underwent biopsy in place of resection). When assembling the control group, the investigators were blinded to survival outcome. RESULTS Significantly greater overall survival was observed for the group treated by TTFields in addition to standard therapy (p &amp;lt; 0.001; Hazard ratio [HR] 0.21; 95% confidence interval [CI] 0.1–0.45; median survival time STDTh-TTF 31.7 months, STDTh 7.1 months). The groups were balanced with respect to sex, and no differences with respect to age (p = 0.13; STDTh-TTF mean 51.58 years, SD 8.8; STDTh mean 52.42 years, SD 8.7) or inclusion date (p = 0.22) by paired t-test were detected. CONCLUSIONS Our initial results appear promising with respect to overall survival benefit in patients undergoing TTFields treatment in addition to standard therapy.

Co-Targeting of CD38 and CD47 in T Cell Acute Lymphoblastic Leukemia

Antibody application is a promising therapy in hematological malignancies including acute lymphoblastic leukemia (ALL). Unlike for B-cell precursor (BCP-ALL), immunotherapeutic interventions in T-cell ALL (T-ALL) are practically non-existent. Most T-ALL patient samples show substantial surface expression of CD38. Moreover, mice bearing T-ALL patient-derived xenograft (PDX) samples treated with daratumumab (Dara) monotherapy displayed prolonged survival and MRD-negativity in 50% of cases as opposed to animals treated with chemotherapy (Vogiatzi et al., Blood, 2019). Besides CD38, elevated surface expression of CD47 has been described in T-ALL (Chao et al., 2011). CD47 acts as a "don't-eat-me" signal protecting cancer cells from macrophage-dependent phagocytosis. In this study, we explored the efficacy of Dara and a CD47 blocking antibody (Fc-modified version of Hu5F9-G4, termed Hu5F9-IgG2σ) alone or in combination in T-ALL. In vitro phagocytosis assays with M-CSF derived M0 (CD64+, CD80-, CD163+) macrophages from healthy donors were evaluated in T-ALL cell lines. Cells labelled with a pH-dependent dye became fluorescent upon engulfment by macrophages and formation of the acidic phagolysosome, which was automatically measured as an absolute cell count. The highest count was set as 100% in each experiment and all other values expressed as percentages relative to that. Treatment of MOLT-13 cells with Dara or Hu5F9-IgG2σ increased median phagocytosis from 5% in control to 23% and 27%, respectively (p=0.008 for both, Figure 1A). However, combined treatment resulted in a maximal increase of median phagocytosis in MOLT-13 cells (p=0.004 compared to Dara and p=0.008 compared to Hu5F9-IgG2σ, Figure 1A). Similar results were obtained in HSB-2 and P-12 cells (p=0.037/0.004 for HSB-2, p=0.011/0.001 for P-12). In addition, phagocytosis was determined in 12 PDX samples from random T-ALL patients. Hu5F9-IgG2σ alone showed a modest increase in median phagocytosis compared to control (19% vs. 6%, p&amp;lt;0.001) and Dara increased phagocytosis to 43% (p&amp;lt;0.001). Combination therapy, however, led to a maximal rise of median phagocytosis in all samples (p&amp;lt;0.001 compared to all others). Phagocytosis was also determined in 16 relapsed/refractory (r/r) T-ALL-PDX samples, in which a maximal median phagocytosis upon combination of Dara and Hu5F9-IgG2σ was also observed (2% in control, 37% in Dara and 14% in Hu5F9-IgG2σ, p&amp;lt;0.001/p=0.02/p=0.02, respectively). The extent of phagocytosis correlated with CD38 and CD47 surface expression on T-ALL cells and, in part, also with expression of signal regulatory protein α (SIRPα) on macrophages, the interacting partner of CD47. A maximal increase in phagocytosis was also observed when applying Hu5F9-IgG2σ in 1/10 of the concentration of Dara, confirming its high efficacy. The effects of the Dara/ Hu5F9-IgG2σ combination were also examined in vivo in phase II-like preclinical trials containing different patients in one experiment. Six random T-ALL-PDX samples were injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and subjected to therapy with Dara and Hu5F9-IgG2σ alone or in combination. In order to mimic minimal residual disease (MRD), antibody therapy was started one day post injection. Mice subjected to Dara displayed prolonged survival in 4/6 (67%) animals compared to control (p=0.007), whereas animals treated with Hu5F9-IgG2σ showed significantly prolonged survival in all cases (p=0.007, Figure 1B). Interestingly, mice not responding to Dara benefitted from Hu5F9-IgG2σ monotherapy and the combination had no further survival advantage in this MRD model. In a second approach, eight r/r T-ALL PDX samples were used, and the same therapy was started upon overt leukemia (1% human blasts in peripheral blood). Compared to control, mice treated with Dara in this overt leukemia and r/r setting showed no survival benefit, while animals subjected to Hu5F9-IgG2σ showed a trend towards a prolonged survival (5/8 cases, 63%, p=0.08). Most importantly, the combination of both agents resulted in a statistically significant survival prolongation in 7/8 cases (88%, p=0.010, Figure 1C). Altogether, we show that combining Dara with CD47 blockade increases phagocytosis in vitro in three T-ALL cell lines and 28 PDX samples. Our in vivo data suggest that this combination is a highly promising therapeutic strategy, especially in the relapsed/refractory setting. Figure Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Kulozik:bluebird bio, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Bourquin:Servier: Other: Travel Support. Schewe:Sobi: Other: was an advisory board member; Bayer: Other: was an advisory board member; Jazz: Other: was an advisory board member; OSE pharmaceuticals: Research Funding.

Abstract IA23: Impact of metastatic prostate cancer on human bone marrow

Abstract Cancer-related mortality due to solid tumor malignancies is overwhelmingly due to the development and progression of metastases. In advanced prostate cancer, metastases most often involve the bone and generally represent incurable disease. It remains unclear what aspects of the bone marrow microenvironment make it hospitable to metastatic dissemination. Similarly, the impact of the metastatic tumor on hematopoiesis and the marrow immune response is poorly understood. We took advantage of rare spinal cord decompression surgeries to profile marrow and metastatic tumors from men with advanced prostate cancer at single-cell resolution. Our analysis contrasts the cellular composition and transcriptional states in matched samples of tumor and liquid bone marrow collected at adjacent vertebral body levels, as well as bone marrow of orthopedic patients without malignancy. Metastatic prostate cancer was associated with hematopoietic suppression and multifaceted immune distortion. There was exhaustion of specific T cell subsets, appearance of inflammatory monocytes and macrophages, and alteration of cytokine profiles. Computational analysis showed association between the presence of specific myeloid subsets and the level of T lymphocyte dysfunction in the tumor fraction. We screened for potential signaling axes that may underlie this interaction. Among them was chemokine CCL20, notably overexpressed by myeloid cells, as was its cognate CCR6 receptor, expressed on T cells. We developed a syngeneic mouse model of bone-metastatic prostate cancer to explore this observation, and demonstrated that disruption of the CCL20-CCR6 axis from either side resulted in significant prolongation of survival. Our results further indicated that this dual overexpression was associated with repressed immune responses. Overall, comparative high-resolution analysis of bone marrow reveals distinct alterations associated with prostate cancer bone metastases that may be amenable to therapeutic targeting with the goal of altering cancer progression. Citation Format: Ninib Baryawno, Youmna Kfoury, Nicolas Severe, Shenglin Mei, Karin Gustafsson, Taghreed Hirz, Thomas Brouse, Elizabeth W. Scadden, Anna A. Igolkina, Bryan D. Choi, Nikolas Barkas, John H. Shin, Philip J. Saylor, David T. Scadden, David B. Sykes, Peter V. Kharchenko, as part of the Boston Bone Metastasis Consortium. Impact of metastatic prostate cancer on human bone marrow [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA23.

Treosulfan-Based Conditioning Regimen for Second Allograft in Patients with Myelofibrosis.

Simple SummaryCurrently, the only curative therapy in myelofibrosis is allogeneic hematopoietic stem cell transplantation. Donor lymphocyte infusion and second stem cell transplantation are the two main treatment options for myelofibrosis patients who relapse after the first transplantation. The optimal conditioning regimen for the second transplantation in myelofibrosis patients is not well defined. Our study aimed to address this question and showed that treosulfan-based conditioning for second allograft in relapsed myelofibrosis patients resulted in longtime freedom from disease in about 50% of the patients. This data supports the second allogeneic hematopoietic stem cell transplantation with a less toxic treosulfan-based conditioning regimen that is effective in relapsed, donor lymphocyte infusion resistant myelofibrosis patients with long term low transplant-related mortality and relapse rates.Relapse after allogeneic hematopoietic stem cell transplantation (AHSCT) in myelofibrosis (MF) patients remains as a significant issue despite advances in transplantation procedures and significant prolongation in survival. Second AHSCT is a potential treatment option but associated with high treatment-related mortality and novel less toxic conditioning regimens are needed. In 33 MF patients with relapse after AHSCT and failure to donor lymphocyte infusion (DLI) we investigated treosulfan (36–42 g/m2) in combination with fludarabine and anti-thymocyte globulin (ATG) as conditioning regimen for a second AHSCT with matched related (n = 2), unrelated (n = 23), or mismatched unrelated (n = 8) donors. All patients achieved leukocyte engraftment after a median of 11 days, and 56 ± 13% experienced acute GVHD grade II–IV at day 100. The therapy-related mortality at day 100 and at 3 years was 16% and 31%, respectively. The cumulative incidence of relapse at 5 years was 16%, resulting in a 5-year disease-free and overall survival of 45% and 47%, respectively. Treosulfan-based conditioning for second allograft in relapsed MF patients resulted in about 50% of the patients in long-term freedom from disease.

Identification of a Novel miR-146a-POU3F2/SMARCA5 Pathway in Glioblastoma

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance primarily contribute to an inevitable tumor recurrence and dismal patient outcome in glioblastoma. Although GBM stem cells (GSCs) are shown to play key roles in these processes, the molecular pathways driving the GSC phenotype (GBM-stemness) remain poorly defined. A global miRNA expression analysis was performed using FFPE GBM tumor samples, and we found that low expression of miR-146a correlated with poor clinical outcome and tumor recurrence. Based on this finding, the hypothesis is that miR-146a functions as a tumor suppressor and inhibits stemness in GBM. Expression analysis was performed using FFPE GBM tumor samples (n = 268) followed by univariable (UVA) and multivariable (MVA) analyses. Functional studies were conducted to define the role of miR-146a-POU3F2/SMARCA5 pathway in GBM tumorigenesis and therapeutic response. MicroRNA-146a was one of the top miRNAs that correlated with better prognosis in GBM patients by UVA analyses (p = 9.21E-05), which was independent of MGMT promoter methylation by MVA analyses (p<0.001). miR-146a expression was significantly downregulated in recurrent GBM tumors compared with the paired primary GBM tumors (p = 0.003). Overexpression of miR-146a significantly inhibited tumor cell growth and sensitized patient-derived primary GBM cells to temozolomide (TMZ) treatment in vitro, and showed statistically significant smaller tumor size (p<0.01) and prolonged survival (p = 0.001) in vivo. In addition, miR-146a was downregulated in glioma cancer stem cells, and overexpression of miR-146a significantly reduced stemness. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Moreover, overexpression of miR-146a significantly inhibited NF-κB pathways through targeting POU3F2 and SMARCA5 in GBM. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. Fundings: R01CA188228 (to A.C., R.B., K.L., and J.S.B-S.), and the T&P Bohnenn Fund for Neuro-Oncology Research (to P.A.R.). R01CA169368, R01CA11522358, R01CA1145128, R01CA108633, 1RC2CA148190, and U10CA180850-01; A Brain Tumor Funders Collaborative Grant; Ohio State University Comprehensive Cancer Center Award (all to A.C.).

Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST= 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

Abstract 6705: An integrated in vitro and in vivo approach providing insights in immune modulation

Abstract In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic's interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p&amp;lt; 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic. Citation Format: Shilina Roman, Julia Schüler, Sanne Holt, Jeroen DeGroot, Omar Aziz, Ian Waddell. An integrated in vitro and in vivo approach providing insights in immune modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6705.

Abstract 4169: The novel thioredoxin reductase inhibitor A-Z2 triggers intrinsic apoptosis and shows efficacy in the treatment of acute myeloid leukemia

Abstract Chemoresistance and high incidence of relapse in acute myeloid leukemia (AML) patients are associated with thioredoxin (Trx) overexpression. Thus, targeting the Trx system has emerged as a promising approach to treating AML. Both arsenicals and azelaic acid (AZA) are thioredoxin reductase inhibitors (TrxR) and possess antileukemic effects. In this study, to exploit agents with higher potency and lower toxicity, we got some organic arsenicals and further synthesized a series of targeted compounds by binding AZA to organic arsenicals, and then screened the most effective one, N-(4-(1, 3, 2-dithiarsinan-2-yl) phenyl)-azelamide (A-Z2). A-Z2 showed a stronger inhibitory effect against TrxR activity and in AML cell lines than did AZA or arsenicals. Additionally, A-Z2 was less toxic to healthy cells compared with traditional chemotherapeutic drugs. A-Z2 induces apoptosis by collapsing of mitochondrial membrane potential, reducing ATP level, releasing of cytochrome c and TNF-α, activating of caspase 9, 8 and 3. Analysis of the mechanism revealed that A-Z2 activates the intrinsic apoptotic pathway by directly selectively targeting TrxR/Trx and indirectly inhibiting NF-κB. A-Z2's better efficacy and safety profile against arsenicals and azelaic acid were also evident in vivo. A-Z2 had better plasma stability and biological activity in rats. A-Z2-treated mice displayed significant symptom relief and prolonged survival in a patient-derived xenograft (PDX) AML model. Herein, our study provides a novel antitumor candidate and approach for treating AML. Keywords: Acute myeloid leukemia, Thioredoxin reductase, TrxR inhibitor, organic arsenicals, NF-κB Citation Format: Fuling Zhou, Dongdong Zhang. The novel thioredoxin reductase inhibitor A-Z2 triggers intrinsic apoptosis and shows efficacy in the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4169.

Abstract 2038: Overall survival of glioblastoma patients treated by standard therapy in comparison to standard therapy plus tumor treating fields

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common malignant primary intracranial tumor and traditionally has a median survival of only 10 to 14 months, with only 3 to 5% of patients surviving more than three years. Recurrence is nearly universal, and further decreases the median survival to only 5 to 7 months with optimal therapy. Tumor treating fields (TTFields; Optune, Novocure, Haifa, Israel) therapy is a novel treatment technique that has recently shown significant prolonged survival in GBM patients. This therapy is approved for the treatment of newly diagnosed and recurrent GBM and is based on the principle that low intensity, intermediate frequency alternating electric fields (100 to 300 kHz) have an anti-mitotic effect in specific cell types. The applied fields disrupt the mitotic spindle, microtubule assembly and the segregation of intracellular organelles during cell division, leading to apoptosis or mitotic arrest. We aimed to compare overall survival between patients treated with standard therapy and standard therapy plus TTFields at our institution. Materials and Methods: A total of 34 patients (22 male, 15 female) diagnosed with GBM and treated by standard therapy plus TTFields (STDTh-TTF) at our institution were included. Standard therapy (STDTh) consisted of surgical resection, followed by combined radiotherapy and chemotherapy (Temozolomide); in 2 cases biopsy was performed in place of resection. The date of resection or biopsy was considered the entry date and was used in calculating survival, and ranged from June 2015 to July 2019. A matching control group treated by STDTh alone at our institution was assembled from our database, based primarily on date of resection or biopsy, and secondarily by age (1 subject underwent biopsy in place of resection). When assembling the control group, the investigators were blinded to survival outcome. Results: Significantly greater overall survival was observed for the group treated by TTFields in addition to standard therapy (p = 0.005; Hazard ratio [HR] 0.28; 95% confidence interval [CI] 0.11-0.69). The groups were identical with respect to sex, and no differences with respect to age (p=0.12; STDTh-TTF mean 50.54 years, SD 10.3; STDTh mean 54.39 years, SD 9.3) or inclusion date (p=0.3) were detected. Conclusions: Our initial results appear promising with respect to overall survival in patients undergoing TTFields treatment in addition to standard therapy. Citation Format: Aaron Rulseh, Adam Derner, Jan Sroubek, Jan Klener, Josef Vymazal. Overall survival of glioblastoma patients treated by standard therapy in comparison to standard therapy plus tumor treating fields [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2038.

Enhanced Anti-Brain Metastasis from Non-Small Cell Lung Cancer of Osimertinib and Doxorubicin Co-Delivery Targeted Nanocarrier.

PurposeCurrently, the treatment of brain metastases from non-small cell lung cancer (NSCLC) is rather difficult in the clinic. A combination of small molecule-targeted drug and chemo-drug is a promising therapeutic strategy for the treatment of NSCLC brain metastases. But the efficacy of this combination therapy is not satisfactory due to the blood–brain barrier (BBB). Therefore, it is urgent to develop a drug delivery system to enhance the synergistic therapeutic effects of small molecule–targeted drug and chemo-drug for the treatment of NSCLC brain metastases.MethodsT7 peptide installed and osimertinib (AZD9291) loaded intracellular glutathione (GSH) responsive doxorubicin prodrug self-assembly nanocarriers (T7-DSNPs/9291) have been developed as a targeted co-delivery system to enhance the combined therapeutic effect on brain metastases from NSCLC. In vitro cell experiments, including intracellular uptake assay, in vitro BBB transportation, and MTT assay were used to demonstrate the efficacy of T7-DSNPs/9291 in NSCLC brain metastasis in vitro. Real-time fluorescence imaging analysis, magnetic resonance imaging analysis, and Kaplan–Meier survival curves were used to study the effect of T7-DSNPs/9291 on an animal model in vivo.ResultsT7-DSNPs/9291 could significantly enhance BBB penetration of AZD9291 and doxorubicin via transferrin receptor-mediated transcytosis. Moreover, T7-DSNPs/9291 showed significant anti-NSCLC brain metastasis effect and prolonged median survival of an intracranial NSCLC brain metastasis animal model.ConclusionT7-DSNPs/9291 is a potential drug delivery system for the combined therapy of brain metastasis from NSCLC.