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Supplemental Figures 1-9. Figure S1. Carbon-Nitride Dot Synthesis. Figure S2. Characterization of CND-Dox-TF. Figure S3. CND cytotoxicity. Figure S4. Cell surface TFR1 expression and receptor recycling. Figure S5. Rapid nuclear entry by Dox after CND-Dox-TF treatment in DLBCL. Figure S6. HEK293 is a model cell line for elucidating CND-Dox-TF mechanism. Figure S7. CND-Dox-TF working dose identified. Figure S8. CND-Dox-TF has improved toxicity profile compared to Dox. Figure S9. R-nanoCHOP treatment has favorable toxicity profile in non-malignant organs.

New treatments are needed to address persistent unmet clinical needs for diffuse large B-cell lymphoma (DLBCL). Overexpression of transferrin receptor 1 (TFR1) is common across cancer and permits cell-surface targeting of specific therapies in preclinical and clinical studies of various solid tumors. Here, we developed novel nanocarrier delivery of chemotherapy via TFR1-mediated endocytosis, assessing this target for the first time in DLBCL. Analysis of published datasets showed novel association of increased TFR1 expression with high-risk DLBCL cases. Carbon–nitride dots (CND) are emerging nanoparticles with excellent in vivo stability and distribution and are adaptable to covalent conjugation with multiple substrates. In vitro, linking doxorubicin (Dox) and transferrin (TF) to CND (CND–Dox–TF, CDT) was 10–100 times more potent than Dox against DLBCL cell lines. Gain- and loss-of-function studies and fluorescent confocal microscopy confirmed dependence of these effects on TFR1-mediated endocytosis. In contrast with previous therapeutics directly linking Dox and TF, cytotoxicity of CDT resulted from nuclear entry by Dox, promoting double-stranded DNA breaks and apoptosis. CDT proved safe to administer in vivo, and when incorporated into standard frontline chemoimmunotherapy in place of Dox, it improved overall survival by controlling patient-derived xenograft tumors with greatly reduced host toxicities. Nanocarrier-mediated Dox delivery to cell-surface TFR1, therefore, warrants optimization as a potential new therapeutic option in DLBCL.Significance:Targeted nanoparticle delivery of doxorubicin chemotherapy via the TRF1 receptor presents a new opportunity against high-risk DLBCL tumors using potency and precision.

Background: Chemotherapy remains the mainstay of treatment for early-stage triple negative breast cancer (TNBC), yet targetable drivers of interest are under investigation. A subset of TNBCs express the androgen receptor (AR) and exhibit androgen-dependent growth. The AR-antagonist enzalutamide (ENZA) has shown activity in patients with metastatic AR+ TNBC. In this study, the feasibility of adjuvant ENZA in early-stage, AR+ TNBC was assessed (NCT02750358). As reported previously, this study met its primary endpoint of feasibility (Traina et al., ASCO 2019). Here we report secondary survival endpoints. Methods: In this single-arm, open-label, multi-center trial, patients with stage I-III, AR≥1% TNBC (ER/PR Citation Format: Elaine M Walsh, Ayca Gucalp, Sujata Patil, Marcia Edelweiss, Dara S Ross, Pedram Razavi, Shanu Modi, Neil M Iyengar, Rachel Sanford, Tiffany Troso-Sandoval, Mila Gorsky, Jackie Bromberg, Pamela Drullinsky, Diana Lake, Serena Wong, Patricia DeFusco, Nicholas Lamparella, Ranja Gupta, Tasmila Tabassum, Leigh Ann Boyle, Artavazd Arumov, Tiffany A Traina. Adjuvant enzalutamide for the treatment of early-stage androgen-receptor positive, triple negative breast cancer: A feasibility study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-14-03.

Chemotherapy remains the most widely used cancer treatment modality. Nanotechnology provides exciting opportunities to improve these drugs, transforming decades-old generic treatments into precise new medicines. We illustrate the potential of recent advances in nanotechnology-enhanced therapy focusing on diffuse large B-cell lymphoma (DLBCL); the most common hematologic malignancy.

New treatments are needed to address persistent unmet clinical needs for diffuse large B-cell lymphoma (DLBCL). Overexpression of transferrin receptor 1 (TFR1) is common across cancer and permits cell-surface targeting of specific therapies in preclinical and clinical studies of various solid tumors. Here, we developed novel nanocarrier delivery of chemotherapy via TFR1-mediated endocytosis, assessing this target for the first time in DLBCL. Analysis of published datasets showed novel association of increased TFR1 expression with high-risk DLBCL cases. Carbon–nitride dots (CND) are emerging nanoparticles with excellent in vivo stability and distribution and are adaptable to covalent conjugation with multiple substrates. In vitro, linking doxorubicin (Dox) and transferrin (TF) to CND (CND–Dox–TF, CDT) was 10–100 times more potent than Dox against DLBCL cell lines. Gain- and loss-of-function studies and fluorescent confocal microscopy confirmed dependence of these effects on TFR1-mediated endocytosis. In contrast with previous therapeutics directly linking Dox and TF, cytotoxicity of CDT resulted from nuclear entry by Dox, promoting double-stranded DNA breaks and apoptosis. CDT proved safe to administer in vivo, and when incorporated into standard frontline chemoimmunotherapy in place of Dox, it improved overall survival by controlling patient-derived xenograft tumors with greatly reduced host toxicities. Nanocarrier-mediated Dox delivery to cell-surface TFR1, therefore, warrants optimization as a potential new therapeutic option in DLBCL. Significance: Targeted nanoparticle delivery of doxorubicin chemotherapy via the TRF1 receptor presents a new opportunity against high-risk DLBCL tumors using potency and precision.

Chemotherapy with or without immunotherapy remains the mainstay of treatment for triple-negative breast cancer (TNBC). A subset of TNBCs express the androgen receptor (AR), representing a potential new therapeutic target. This study assessed the feasibility of adjuvant enzalutamide, an AR antagonist, in early-stage, AR-positive (AR +) TNBC.This study was a single-arm, open-label, multicenter trial in which patients with stage I-III, AR ≥ 1% TNBC who had completed standard-of-care therapy were treated with enzalutamide 160 mg/day orally for 1 year. The primary objective of this study was to evaluate the feasibility of 1 year of adjuvant enzalutamide, defined as the treatment discontinuation rate of enzalutamide due to toxicity, withdrawal of consent, or other events related to tolerability. Secondary endpoints included disease-free survival (DFS), overall survival (OS), safety, and genomic features of recurrent tumors.Fifty patients were enrolled in this study. Thirty-five patients completed 1 year of therapy, thereby meeting the prespecified trial endpoint for feasibility. Thirty-two patients elected to continue with an optional second year of treatment. Grade ≥ 3 treatment-related adverse events were uncommon. The 1-year, 2-year, and 3-year DFS were 94%, 92% , and 80%, respectively. Median OS has not been reached.This clinical trial demonstrates that adjuvant enzalutamide is a feasible and well-tolerated regimen in patients with an early-stage AR + TNBC. Randomized trials in the metastatic setting may inform patient selection through biomarker development; longer follow-up is needed to determine the effect of anti-androgens on DFS and OS in this patient population.

BACKGROUND: A subset of TNBC expresses a luminal signature and demonstrates AR-dependence. Enzalutamide (ENZA), an AR-antagonist, has activity with metastatic AR+ TNBC, with a clinical benefit rate of 33% (Traina et al, JCO 2018). We recently demonstrated that one year (y) of adjuvant ENZA is feasible in early stage, AR+ TNBC, with no new safety signals observed (Traina ASCO 2019). Fatigue was the most common, treatment (tx)-related, adverse event of any grade according to investigator assessment (24%). Here we report per-protocol planned secondary analyses of PROs during one year of ENZA including quality of life (QoL), fatigue, and self-reported physical activity (NCT02750358). METHODS: Eligible patients have Stage I-III, ER/PR RESULTS: Between 5/2016 and 6/2018, 50 patients were enrolled. As of 6/27/19, 34 patients completed 1y of ENZA; 15 patients are off tx: recurrence (3), toxicity (5), nonadherence (4), and consent withdrawal (3). Patients evaluable for PRO (N): baseline (50), 12 weeks (43) and 52 weeks (34). Median scores on FACT-B and fatigue scales remained stable over the course of adjuvant ENZA in the 1st year of therapy (Table). Self-reported exercise increased during this period. Table. Median scores (range) for PROs during 1 year of adjuvant ENZATime PointBaselineWeek 12Week 52FACT-G88.0 (53.5-105.0)82.0 (36.4-105.0)89.5 (58.0-105.0)Breast Cancer Subscale28.5 (15.6-39.0)29.0 (9.0-40.0)29.0 (19.0-40.0)Total118.0 (77.0-141.0)111.0 (45.4-145.0)121.0 (83.0-145.0)FACT-B trial outcome index73.0 (43.0-92.0)73.0 (25.7-96.0)76.9 (51.0-96.0)FACIT43.0 (17.0-52.0)39.0 (10.0-52.0)43.0 (11.0-52.0)Total Exercise (min/week)120 (0-1260)152 (0-3120)180 (0-2580) CONCLUSIONS: Patient reported outcomes of health related QOL remain stable when ENZA is administered for 1 year in the adjuvant setting. Self-reported time spent engaged in physical exercise increased numerically during 1y of therapy. During presentation, additional analyses will be reported including QOL at additional time points, relationship of FACT-B score with FACIT fatigue score and exercise, and QOL changes in relation to relative dose intensity of ENZA, toxicity and off study reason. Citation Format: Tiffany A Traina, Lee W Jones, Victoria Blinder, Jessica Scott, Leigh Ann Boyle, Artavazd Arumov, Tina Alano, Sujata Patil, Patricia DeFusco, Nicholas Lamparella, Mark Robson, Ayca Gucalp. Patient-reported outcomes (PROs) during one year of adjuvant enzalutamide for the treatment of early stage androgen receptor positive (AR+) triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-12-09.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

Effector lymphocytes are multifunctional cells of the immune system that promote cytolysis of pathogen-infected cells and nascent tumors. Tumors must learn to evade effectors and employ a wide variety of mechanisms to do so. Bispecific Abs (BsAbs) are an emerging cancer immunotherapy approach seeking to re-engage either T effectors or NK cells with malignant cells. Possessing specificity for effector cells on one end and a tumor Ag on the other, these molecules work by attracting effectors to the target cell to build an immunologic synapse and induce tumor cell killing. The BsAb blinatumomab, for example, has specificity for the T cell–activating cell surface protein CD3 and the B cell Ag CD19. The only BsAb with regulatory approval currently, blinatumomab is used in the treatment of relapsed or refractory B cell acute lymphoblastic leukemia. Many additional BsAbs are in preclinical development, however, targeting many different tumor types. The variety of potential effector cells and cancer Ags, along with potential combination therapies, make BsAbs an active area of drug development. In this review, we discuss cancer recognition by the immune system and structural and mechanistic aspects of BsAbs. We summarize key steps in preclinical development and subsequent translation to medical practice. Future directions for BsAbs include combinations with a wide variety of both immunologic and nonimmunologic therapies. Defining their optimum clinical use is at early stages.

3120 Background: The cell-surface transferrin receptor TFR1 imports iron-bound transferrin into cells via clathrin-mediated endocytosis. Tumors require constitutive iron import to drive proliferation, and several studies establish TFR1 as a target able to facilitate intracellular delivery of cytotoxic therapeutic molecules. Our own work previously revealed association between high expression of TFRC, the gene encoding TFR1, and high risk for poor outcome in diffuse large B-cell lymphoma (DLBCL). We showed therapetuic targeting of TFR1 in DLBCL results in significant anti-tumor benefit. Systematic analysis of TFRC expression as a prognostic marker across tumor types, however, has not been investigated. Methods: Tissue samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. Overall survival (OS) was calculated from date of tissue collection to last contact from insurance claims data and employed Kaplan-Meier analysis by Wilcoxon statistics, with p < 0.05 defined as significant. Results: Amongst 47 cancer types included, colorectal cancer (CRC) displayed the highest level of TFRC mRNA, followed by gastric cancer. In an all-tumor cohort (n = 93248), patients with higher TFRC expression (cutoff = median) had significantly worse OS (HR = 1.348, 95% CI [1.317-1.38], p < 0.00001). This was statistically significant in 23 individual tumor types. Drilling down further, TFRC adverse prognostic value was mainly driven by cohorts with larger number of samples in the database, including non-small cell lung cancer (n = 17309), CRC (n = 12860), breast cancer (n = 8632), ovarian carcinoma (n = 7998), uterine neoplasms (n = 6097), prostate adenocarcinoma (n = 3411), glioblastoma (n = 2821), gastric cancer (n = 1579), and others. Surprisingly, TFRC overexpression correlated with improved outcome in vulvar squamous cell carcinoma (VSCC, n = 297). TFRC was found to be most prognostic in prostate adenocarcinoma with median OS 1139 days in pts with high vs 3230 days in pts with low TFRC (HR = 2.556, 95% CI [2.213-2.951], p < 0.00001). Conclusions: Our study is the first to combine modern molecular profiling with a large cohort of clinical tissue samples to reveal a prognostic role for TFRC expression in a variety of solid tumor types. We found TFRC overexpression to be prognostic in a large proportion of histologies, though surprisingly association with improved OS in VSCC. Highest expression occured in CRC and gastric cancer, diseases with needs for new therapies. A number of TFR1-targeting therapeutics are currently at various stages of development, and warrant further investigation in disease cohorts identified from our study.

Background: The androgen-signaling pathway plays a role in breast cancer (BC) pathogenesis and emerging evidence suggests the androgen receptor (AR) is a therapeutic target. TBCRC011 established safety and efficacy of inhibiting AR with bicalutamide (B) in patients (pts) with androgen AR+/ER/PgR- metastatic BC (MBC). Treatment with B at 150 mg oral daily demonstrated a clinical benefit rate (CBR) of 19% in this population. The rationale for combining palbociclib (P) with AR blockade stems from prior work in ER+ MBC. P is an oral, selective inhibitor of CDK4/6 activity which significantly improved median progression-free survival (PFS) in combination with letrozole compared to letrozole monotherapy for the treatment of postmenopausal pts with ER+ MBC in the first-line setting. Consistent with preclinical data, P has been shown to reduce growth of AR+ ER/PgR- MDA-MB-453 breast cancer cells via reduced Rb phosphorylation. It has been shown that AR+ TNBC expresses a luminal profile and has intact Rb protein, the target of palbociclib activity. Therefore, we hypothesize that P will increase the efficacy of B in pts with metastatic AR+ TNBC. NCT02605486. Methods: Pts with AR+ (IHC ≥ 1%)/ER any/HER2(-) MBC on central review at MSK were eligible if met following criteria: ECOG ≤2, postmenopausal, no limit to prior regimens. Pts with ER+ BC must have had 1 prior endocrine therapy. Treatment: B orally daily and P orally daily 3 weeks on 1 week off. DLT period = 28 days. Pts are evaluated for toxicity every 2-4 weeks and for response every 8 weeks. Phase I standard 3+3 design with 3 dose escalations. The primary objective of the phase I portion of the study is to determine the recommended phase II dose of P in combination with B. Plasma for pharmacokinetics (PK) was collected throughout the study. Results: As of 5/11/17, 15 pts with AR+ MBC were enrolled to the phase I portion. Target accrual has been met for the phase I and has since closed. No DLTs were reported in any of the dose escalation cohorts. The maximum tolerated dose was determined to be B 150 mg daily and P 125 mg daily for 21 days in a 28 day cycle. Treatment has been well tolerated with no related Grade 4 or 5 adverse events (AEs). The most common grade 3 significant AEs were hematologic including neutropenia n =5, leukopenia n= 4, and lymphocytopenia n=3 and thought to be related to P. One Grade 4 hypercalcemia led to hospitalization and was deemed related to disease progression. Median time on study was 8 weeks (2-47 weeks). One patient from the phase I remains on study. PK analysis is ongoing and will be presented, including drug-drug interaction data. Conclusions: The combination of B+P has been well tolerated with no unexpected toxicity observed. Updated safety, response, and PK data will be presented. Enrollment on phase II is ongoing. Citation Format: Gucalp A, Edelweiss M, Patil S, Gounder MM, Feigin KN, Corben A, Arumov A, Traina TA. Phase I/II trial of palbociclib in combination with bicalutamide for the treatment of androgen receptor (AR)+ metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-11-04.

SUMMARY Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple “translational inhibition” as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical “translation inhibitors” is mediated by comprehensive translational landscape remodeling., Graphical abstract, In brief Tactical protein synthesis inhibition is actively pursued as a cancer therapy that bypasses signaling redundancies limiting current strategies. Ho et al. show that rocaglates, first identified as inhibitors of eIF4A activity, globally reprogram cellular translation at both protein synthesis machinery and translatome levels, inducing cytotoxicity through anti-survival GEF-H1/RHOA/JNK signaling.

Rocaglates are under active development for cancer therapy, with the first-in-class member zotatifin recently entering Phase I/II clinical trial for advanced solid tumor malignancies (NCT04092673). Initiation of this trial was based purely on rocaglates' inhibitory activity against translation initiation factor and RNA helicase eIF4A. Rapid development of these compounds towards clinical use urgently requires a complete understanding of their mechanisms. Critically, however, the full effects of rocaglates on protein synthesis are unknown, especially with regards to up-regulated proteins and rocaglate-activated translation factors that mediate their potent anticancer activity. Past rocaglate studies focused exclusively on mechanisms and targets of translational inhibition, relying exclusively on indirect RNA-based approaches that do not reliably capture proteome-level changes, a caveat proven in cancer cells responding to drugs. Formal proteomic investigations of rocaglate effects are non-existent in the literature. We addressed these vital knowledge gaps in this study. An original proteome-level analysis using metabolic pulse-labeling and TMT-mass spectrometry revealed extensive rocaglate-induced changes in protein output (translatome), including myriad rocaglate-up-regulated proteins that drive cytotoxicity through currently uncharacterized mechanisms. Specifically, we find in vitro and in vivo that rocaglates, including zotatifin, induce GEF-H1, activating anti-survival RHOA/JNK signaling. Intriguingly, these responses occur regardless of eIF4A expression. Beyond translatome remodeling, mass spectrometry-based MATRIX interrogation of the protein synthesis machinery revealed widespread rocaglate-dependent changes. As a prime example, rocaglate-augmented eEF1ϵ1 activity mediates initiator methionine-tRNAiMet delivery to drive synthesis of rocaglate-inducible proteins and phenotypes. Overall, this study transforms the current definition of rocaglates from one-dimensional eIF4A inhibitors to translation remodelers that globally reprogram protein output and the translation machinery. As human patients begin treatment with these compounds, our discovery of rocaglate-inducible proteins and rocaglate-activated translation factors that drive drug-specific responses highlights an entirely uncharacterized yet critical aspect of rocaglate potency. These timely findings emphasize systemic translational modeling as a newer concept for cancer therapy, and advocate for a systematic overhaul in our understanding of drugs traditionally defined as translation inhibitors. Citation Format: Jyun David Ho, Jr, Tyler A. Cunningham, Caroline A. Coughlin, Artavazd Arumov, Evan R. Roberts, Daniel Bilbao, Jonathan R. Krieger, Stephen Lee, Jonathan H. Schatz. Translatome and protein synthesis machinery remodeling by rocaglates drives antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1031.

Diffuse large B-cell lymphoma (DLBCL) comprises a third of non-Hodgkin lymphoma (NHL), with incidence steadily rising in what is already the most common hematologic malignancy. The backbone of potentially curative frontline chemoimmunotherapy combinations like R-CHOP is doxorubicin (Dox), still considered the most active anti-lymphoma agent. Relapsed or refractory (rel/ref) disease after frontline treatment carries persistently poor prognosis, even with recent advances in immunotherapy. Development and implementation of new therapeutic strategies remain urgent priorities to address unmet clinical needs in both high-risk previously untreated and rel/ref patients. We have studied transferrin receptor (TFR1) expression as a marker of high-risk DLBCL revealing significantly worse outcomes following frontline therapy associated with high expression in diagnostic samples. TFR1 is therefore a rational target for treatments aimed specifically at high-risk DLBCL. We used third-generation carbon-nitride dot (CND) nanocarriers we have developed conjugated to Dox and holo-transferrin (TF) to develop a chemotherapeutic-nanocarrier (NanoDox) designed to deliver Dox to TFR1-expressing tumors while sparing non-malignant tissues. Dox's mechanism is primarily through nuclear DNA damage or induction of reactive oxygen species (ROS) H202. We treated DLBCL cell lines with NanoDox or Dox for 24-hours and observed induction of nuclear DNA damage marker γ-H2Ax at drastically lower doses (10 nM NanoDox vs. 100 nM Dox). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results yielded similar findings in BJAB cells, as observed under γ-H2Ax analysis, with TUNEL induction at significantly lower NanoDox concentrations. Treatment with NanoDox did not generate significant changes in ROS H202 when compared to Dox at 24-hours. Dox is known for a strong cytotoxic profile that extends past its terminal half-life of 24-36 hours. Therefore, we treated BJAB, Farage and DHL4 cells for 24-hours with a range of NanoDox and Dox doses, followed by washout and tracked cell viability for 6 additional days. We found as low as 10 nM NanoDox induced rapid and irreversible cytotoxicity, an effect only seen at much higher doses of Dox. These results combined with fluorescent confocal microscopy studies confirmed NanoDox works primarily through rapid nuclear Dox accumulation, causing DNA damage and apoptosis. Building on initial NanoDox dosing studies in NOD Scid Gamma (NSG) mice, we constructed R-nanoCHOP as an alternate to R-CHOP by replacing Dox with NanoDox at a working dose (WD) of 33.0 mg/kg. We next engrafted patient-derived xenograft (PDX) tumors derived from a previously untreated patient with germinal center B-cell (GCB) subtype DLBCL. We randomized NSG animals to two groups at tumor engraftment and treated with 21-day cycles of R-nanoCHOP (n=10) or R-CHOP (n=11). The treatments controlled tumor volume (TV) similarly, but R-nanoCHOP significantly improved overall survival associated with greatly reduced toxicities to host animals, which tolerated an average of 2 additional cycles of R-nanoCHOP. Studies with murine cells in vitro, including A20 B-cell lymphoma cells, confirmed these effects were not due to species differences, with human holo-TF alone and as part of NanoDox binding mouse and human TFR1 with equal affinity. This work establishes proof of principle in DLBCL for targeting TFR1, a specific marker of clinically high-risk disease. CND nanocarriers provide a flexible platform to exploit this, with our initial studies enhancing the safety profile of Dox, which remains the most active frontline DLBCL agent more than 50 years after it entered use. We are now optimizing NanoDox by substituting the holo-TF with an anti-TFR1 single chain variable fragment (scFv), decreasing size of the overall nanoconjugate by >50%. We believe dosing optimization from this change and additional optimizations to Dox delivery intracellularly will permit investigational new drug (IND) studies of absorption, distribution, metabolism and excretion (ADME). In sum, we provide compelling clinically relevant evidence for targeting TFR1 in DLBCL as a new therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy, is an aggressive form of non-Hodgkin lymphoma. Approximately 60% of DLBCL patients achieve long-term disease-free survival from frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), but patients with relapsed or refractory disease have poor prognosis. Doxorubicin (Dox) remains the most active anti-lymphoma agent, but its use is limited by well-characterized toxicities, including potentially irreversible cardiac damage. Targeted and specific delivery of Dox to tumors to overcome these limitations has been an area of active research. Targeted delivery of Dox via the transferrin receptor (TFR1) has preclinical efficacy in solid malignancies but has yet to be explored in lymphoma. We have uncovered for the first time a significant association between TFR1 expression and poor prognosis in DLBCL. Carbon-nitride dots (CNDs) are third-generation nanocarriers with inherent photoluminescence, which are easily conjugated with a wide variety of substrates. We conjugated Dox and transferrin (TF), the ligand for TFR1, to CNDs to develop CND-Dox-TF (CDT). CDT is uniquely designed to enhance Dox delivery to TFR1-expressing DLBCL tumors while limiting effects on nonmalignant tissues. BJAB and Farage cell lines treated with CDT for 24 hours underwent apoptosis at a significantly lower concentration when compared to Dox (50 nM vs. 1000 nM). Overall, CDT was 1-2 Log10 more potent than Dox against DLBCL cell lines. Co-incubation with 250 uM of antagonist holo-transferrin significantly decreased BJAB and Farage sensitivity to CDT, confirming activity through TFR1. Confocal microscopy of BJAB cells incubated up to 24 hours with 250 nM Dox and 30 nM CDT reveled enhanced nuclear colocalization of Dox at a significantly lower concentration. Prior dose-finding experiments in non-tumor bearing mice identified a CDT safe working dose of 33.0 mg/kg containing 16% moles of Dox in comparison to Dox maximum-tolerated dose (MTD) 3.3 mg/kg. We engrafted 2 groups of 10 NSG mice with a Dox-sensitive high-TFR1 expressing patient-derived xenograft (PDX) tumor. Mice were dosed with CDT and Dox on day 0, 14, and 24. CDT demonstrated similar anti-lymphoma efficacy with reduced toxicity: CDT-treated mice displayed no significant decline in body weight, which is characteristic and was observed following each single-agent Dox treatment. Histology analyses revealed little to no obvious damage to CDT-treated nonmalignant tissues, including myocardium. With a working dose of CDT identified, we now have under way clinically relevant R-CHOP vs. R-nanoCHOP (CDT substituted for Dox) assessments in our PDX mouse model. Capitalizing on the unique design, we expect to observe improved anti-lymphoma efficacy, with decreased toxicities and an improvement in overall survival. In sum, we provide mechanistic insight for novel DLBCL nano-chemotherapy and illustrate preclinical efficacy for a promising new therapeutic approach. Citation Format: Artavazd Arumov, Piumi Y. Liyanage, Asaad Trabolsi, Evan R. Roberts, Braulio Ferreira, Daniel Bilbao, Roger M. LeBlanc, Jonathan H. Schatz. Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-48.

1017 Background: Genome-wide transcriptional analysis has identified a unique subset of androgen receptor (AR) +, estrogen receptor (ER)/progesterone receptor (PR)- breast cancer (BC). The functional role of AR was confirmed initially in preclinical models demonstrating that androgen-driven growth could be abrogated by antiandrogen therapy. TBCRC011 established the safety and efficacy of inhibiting AR with bicalutamide (B) in patients (pts) with AR+/ER/PR- metastatic BC (MBC) with a median progression free survival (PFS) of 12 weeks (wks) (95% CI, 11–22 wks). In preclinical data, palbociclib (P) has been shown to reduce growth of AR+/ER/PR- MDA-MB-453 BC cells. It has been shown that AR+ triple negative BC (TNBC) expresses a luminal profile and has intact Rb protein, the target of P activity. We conducted this Phase I/II trial of the AR inhibitor B in combination with the CDK4/6 inhibitor P in pts with AR+/ER/PR/HER2- BC (NCT02605486) to test the hypothesis that androgen blockade, paired with CDK4/6 inhibition would have increased efficacy in pts with androgen-dependent BC. Methods: Postmenopausal pts with AR+ TN MBC defined as IHC ≥ 1% nuclear staining (DAKO, Clone AR441 (5/2016-11/2016) then Ventana AR SP107 (11/2016-6/2018), ECOG ≤2, measurable/non-measurable disease were eligible for enrollment. Any number of prior regimens was permitted. Pts received B 150 mg daily and P 125 mg daily 3 wks on 1 wk off. Pts were evaluated for toxicity every 2-4 wks and for response every 8-12 wks. Primary endpoint: 6 month (mo) PFS. Secondary endpoints: clinical benefit rate, toxicity, correlative studies to better characterize AR+ TNBC. A Simon 2-stage minimax design that discriminates between 6 mo PFS rates of 20% and 40% was used. If ≥ 11/33 pts were PF at 6 mo then B+P would warrant further study. Results: As of 1.1.20 33 pts were enrolled on study with median (med) age 67 (42-79), performance status 0 (0-1). Number of pts with visceral metastases: 20, measurable disease: 22. AR% 1-9: 3, 10-50: 6; 51-100: 24. Med prior lines for MBC: 3 (0-9). Best response: (31 evaluable pts): 11 pts PF at 6mo: 10 SD > 6mo, 1 PR. Med wks on study: 14 (2-101). Toxicity > 10% grade >3 related: Number of pts with leukopenia: 21, neutropenia: 21, lymphocytopenia: 6, thrombocytopenia: 3. One pt with febrile neutropenia. One death due to disease progression within 30 days off study. Conclusions: In this selected subset of pts with AR+ TN MBC, this study met its prespecified endpoint with 11 pts PF at 6 mo on B 150 mg + P 125 mg. B+P has been well tolerated with no unexpected toxicity observed. Clinical trial information: NCT02605486 .

Doxorubicin (Dox) remains the most active drug against aggressive lymphomas, forming the backbone of multiple potentially curative frontline combination regimens. R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), for example, cures diffuse large B-cell Lymphoma (DLBCL), the most common lymphoid malignancy in the United States, greater than 60% of the time. Patients with relapsed/refractory disease, however, have poor prognosis and require new options. Advances in nanotechnology provide new opportunities to widen therapeutic windows for existing drugs by enhancing delivery to tumor cells and limiting toxicities to non-malignant tissues. Carbon-Nitride Dots (CND) are novel nanocarriers we have developed that can be conjugated with a diverse range of molecules and have an established safe pharmacologic profile. Here, we sought CND-based enhancement of Dox's anti-lymphoma activities. We generated unconjugated CNDs (~3 nm) through a hydrothermal microwave synthesis, followed by carbodiimide cross-linking bioconjugation steps to covalently link Dox and/or transferrin (TF), the protein ligand for the transferrin receptor (TFR). Because TFR is expressed on the cell surface in a range of B-cell lymphomas including DLBCL, we aimed to increase Dox delivery to tumor cells and limit delivery to non-malignant tissues. We probed a cohort of DLBCL cell lines for TFR expression via western blot, followed by baseline viability assays. Unconjugated CNDs showed no toxicity in vitro, while conjugation to Dox alone resulted in potency similar to unconjugated Dox. CND-Dox-TF, however, was 1-2 Log10 more potent than Dox alone (LD50 1.2-48.5 nM vs. 205.5->1000 nM), consistent with enhanced activity due to the entry of the nanocarrier into the cells through the TF-TFR interaction. To create a functional model, we cloned the TFR1 gene into a GFP-lentiviral expression vector. We infected previously tested cell lines with TFR1, confirmed increased TFR expression via western blot, and exposed cells to CND nanocarriers. TFR1-infected cells showed selective disadvantage during CND-Dox-TF treatment compared to uninfected and empty-vector controls, while CND and CND-Dox controls showed no differential effect. After establishing CND-Dox-TF proof of principle in vitro, we next initiated testing in vivo, beginning with maximum-tolerated dose (MTD) finding experiments. We treated three groups of non-tumor-bearing NOD scid gamma (NSG) mice intravenously with a single dose of (1) CND, (2) CND-Dox, and (3) CND-Dox-TF. Maximum dosing of CND-Dox-TF based on solubility in 100 µL PBS was roughly 1:4 molar equivalent to Dox MTD (3.3 mg/kg). This dose of CND-Dox-TF caused decreased body weight to >20% loss from starting dosing and animals had to be sacrificed, with organ pathology pending. Molar equivalent dosing of CND and CND-Dox, however, resulted in no weight loss, demonstrating biologic activity of the TF moiety but unfortunately intolerable toxicity at this initial dose. We then reduced the CND-Dox-TF dose to 1:16 molar equivalent to Dox MTD and repeated dosing to three non-tumor-bearing animals. This resulted in decreased body weight of 10% from starting dose by day 11, followed by a rebound to normal body weight by day 17. This observed body weight fluctuation is similar to what is seen under a 0.75% of Dox MTD. With a dose of 33 mg/kg identified as the MTD of the CND-Dox-TF, we are now proceeding to anti-tumor efficacy experiments in four available PDX models of DLBCL (two each from previously untreated and relapsed/refractory patients). We will compare the MTD of Dox versus the MTD of CND-Dox-TF initially as single agents. We will then compare R-CHOP to "R-nanoCHOP" (replacement of Dox with the MTD of CND-Dox-TF). We hypothesize that both through the TF-TFR interaction and enhanced tumor permeability and retention that are known properties of CNDs, CND-Dox-TF treated mice will have improved anti-lymphoma responses. In sum, we show that a TF ligand conjugated to Dox via our CND nanocarrier significantly increases the anti-lymphoma efficacy of Dox on DLBCL cell lines. Importantly, we also show these novel nanocarrier therapeutic molecules are safe to administer in vivo, and we define MTD for studies moving forward. Proof of principle that CND nanotechnology enhances anti-lymphoma activity of Dox would open the door for many such approaches aimed at a variety of malignancies. Disclosures No relevant conflicts of interest to declare.

546 Background: A subset of TNBC is dependent on AR signaling. Enzalutamide (ENZA), an AR-antagonist, has activity in patients (pts) with metastatic AR+ TNBC, with a clinical benefit rate of 33%. This study tests the feasibility of adjuvant ENZA for the treatment (tx) of early stage, AR+ TNBC. We now report the primary endpoint (endpt) and safety. Methods: Eligible pts have centrally confirmed, Stage I-III, ER/PR < 1%, HER2(-), AR ≥1% BC and completed all planned surgery, chemotx and radiation (RT) < 6 months of tx start. AR testing by IHC per MSK methods. Tx consists of ENZA 160mg daily for 1 year (y) with the option to extend tx to 2y. Toxicity per NCI CTCAEv4 every (q) 4 weeks (wk) for 12 wk, then q3 months. Primary endpt: feasibility of 1y ENZA defined as the discontinuation rate due to toxicity, consent withdrawal or tolerability. 50 pts are enrolled to have 46 evaluable pts required to discriminate between feasibility of 50% and 70%, with type I error 5% and 88% power. Pts who have disease progression (PD) or die during 1st y of ENZA and do not have tx discontinuation due to the above will not be included in the primary analysis. If 29 pts complete 1y, adjuvant ENZA will be deemed feasible. Secondary endpts: safety and 3y DFS and OS. Exploratory endpts: PROs and biomarker development. Results: Between 5/2016-6/2018, 50 pts were enrolled. Pt and tumor characteristics (N = 50): Median age 55y (33-81); Stage: I 20 (40%), II 23 (46%), III 7 (14%); Grade (gr): 2 = 26%, 3 = 74%. AR > 10% = 35 (70%), AR ≤10% = 15 (30%). Chemotx 47/50 (94%): Neoadjuvant (neo) 40%, Adjuvant (adj) 60%; Anthracycline/Taxane-based 38/47 (81%), Platinum 1/47 (2%), Docetaxel/Cyclophosphamide 3/47 (6%), other 5/47 (11%). 13/19 who received neo tx failed pCR; 9/13 (69.2%) received adj capecitabine. RT: 38/50 (76%). 27 pts completed 1y of tx. 7 pts will be evaluable by 6/1/19. 1 pt to complete 1y 6/21/19. 15 pts are off tx: PD (3), toxicity (5), noncompliance (4), withdrawal of consent (3). Tx-related AEs, any gr, > 10% (N = 50): fatigue (48%), hot flashes (22%), headache (18%), hyperglycemia (18%), nausea (18%), WBC decreased (16%), dizziness (14%), arthralgia (12%), dyspnea (12%). Tx-related, gr 3 AEs: fatigue (6%), hyperglycemia (2%), hypertension (2%). No gr 4/5 AEs or seizures. 11 pts had dose reduction. Conclusions: Feasibility of adjuvant ENZA will be fully evaluable in 4/2019 and is anticipated to meet the prespecified statistical expectations for primary endpt. ENZA is well tolerated following locoregional tx and standard of care systemic tx. Secondary analyses and correlatives are ongoing to define the role of AR in TNBC. Clinical trial information: NCT02750358.