TRAF2 is an NF-κB activating oncogene in epithelial cancers

Aberrant NF-κB activation is frequently observed in human cancers. Genome characterization efforts have identified genetic alterations in multiple components of the NF-κB pathway, some of which have been shown to be essential for cancer initiation and tumor maintenance. Here using patient tumors and cancer cell lines, we identify the NF-κB regulator, TRAF2 as an oncogene that is recurrently amplified and rearranged in 15% of human epithelial cancers. Suppression of TRAF2 in cancer cells harboring TRAF2 copy number gain inhibits proliferation, NF-κB activation, anchorage-independent growth and tumorigenesis. Cancer cells that are dependent on TRAF2 also require NF-κB for survival. The phosphorylation of TRAF2 at serine 11 is essential for the survival of cancer cells harboring TRAF2 amplification. Together these observations identify TRAF2 as a frequently amplified oncogene

IKK∊-Mediated Tumorigenesis Requires K63-Linked Polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 Ubiquitin Ligase Complex

IκB kinase ∊ (IKK∊, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ∼30% of breast cancers, that promotes malignant transformation through NF-κB activation. Here, we show that IKK∊ is modified and regulated by K63-linked polyubiquitination at lysine 30 and lysine 401. Tumor necrosis factor alpha and interleukin-1β stimulation induces IKK∊ K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKK∊ kinase activity, IKK∊-mediated NF-κB activation, and IKK∊-induced malignant transformation. Disruption of K63-linked ubiquitination of IKK∊ does not affect its overall structure but impairs the recruitment of canonical NF-κB proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKK∊. Altogether, these observations demonstrate that K63-linked polyubiquitination regulates IKK∊ activity in both inflammatory and oncogenic contexts and suggests an alternative approach to targeting this breast cancer oncogene

A Modeled Hydrophobic Domain on the TCL1 Oncoprotein Mediates Association

AKT has a critical role in relaying cell survival and proliferation signals initiated by ligand binding to surface receptors in mammalian cells. Induction of AKT serine/threonine kinase activity is augmented by the T-cell leukemia-1 (TCL1) oncoprotein through a physical association requiring the AKT pleckstrin homology domain. Here, we used molecular modeling and identified an exposed hydrophobic patch composed of two discontinuous amino acid stretches near one end of the TCL1 #-barrel that was required for a TCL1-AKT association. Site-directed mutations of this region did not affect TCL1 secondary structure, yet they disrupted interactions with AKT. This region was found in other members of the TCL1 oncoprotein family, such as TCL1b and MTCP1, and suggested a conserved, novel AKT binding domain. Interestingly, TCL1 and AKT co-localize in multiple cell compartments, but only extracts from the plasma membrane stimulate optimal complex formation in vitro. Identification of an AKT binding domain on TCL1 is an important step in deciphering the complex interactions that regulate AKT kinase activity in lymphocyte development and neoplasia within the immune system

Dysregulated TCL1 requires the germinal center and genome instability for mature B-cell transformation

Most lymphomas arise by transformation of germinal center (GC) B cells. TCL1, a proto-oncogene first recognized for its role in T-cell transformation, also induces GC B-cell malignancies when dysregulated in pEμ-B29-TCL1 transgenic (TCL1-tg) mice. Clonal B-cell lymphomas develop from polyclonal populations with latencies of 4 months or more, suggesting that secondary genetic events are required for full transformation. The goals of this study were to determine the GC-related effects of TCL1 dysregulation that contribute to tumor initiation and to identify companion genetic alterations in tumors that function in disease progression. We report that compared with wild-type (WT) cells, B cells from TCL1-tg mice activated in a manner resembling a T-dependent GC reaction show enhanced resistance to FAS-mediated apoptosis with CD40 stimulation, independent of a B-cell antigen receptor (BCR) rescue signal. Mitogenic stimulation of TCL1-tg B cells also resulted in increased expression of Aicda. These GC-related enhancements in survival and Aicda expression could underlie B-cell transformation. Supporting this notion, no B-cell lymphomas developed for 20 months when TCL1-tg mice were crossed onto an Oct coactivator from B cell (OCA-B)–deficient background to yield mice incapable of forming GCs. Spectral karyotype analyses showed that GC lymphomas from TCL1-tg mice exhibit recurrent chromosome translocations and trisomy 15, with corresponding MYC overexpression. We conclude that pEμ-B29-TCL1 transgenic B cells primed for transformation must experience the GC environment and, for at least some, develop genome instability to become fully malignant

Abstract CT004: Product attributes of axicabtagene ciloleucel (axi-cel) that associate differentially with efficacy and toxicity in second-line large B-cell lymphoma

Abstract Background: Axi-cel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory large B-cell lymphoma after ≥2 lines of systemic therapy. ZUMA-7, a global Phase 3 randomized study, showed superiority of axi-cel over standard second-line therapy (N=359; event-free survival [EFS] HR 0.398, P<.0001; median EFS 8.3 vs 2 months; estimated 2-year EFS 41% vs 16%; objective response rate [ORR] 83% vs 50%, Locke et al. N Engl J Med. 2021). Here we report axi-cel pharmacokinetics (PK), pharmacodynamics (PD), and product attributes associated with ZUMA-7 clinical outcomes. Methods: Samples from patients who received axi-cel (n=170) were analyzed. PK, PD, and axi-cel T-cell composition (naive, CCR7+CD45RA+; differentiated, CCR7-) were assessed for associations with safety and efficacy using previously described methodologies (Neelapu et al. NEJM. 2017; Locke et al. Blood Adv. 2020). Results: The median (Q1, Q3; n=162) peak CAR T-cell level, time to peak, and area under the curve within the first 28 days of treatment (AUC0-28) were 25.8 cells/μL (8.2, 57.9), 8 days (8, 9), and 236.2 cells/μL*days (76.4, 758.0), respectively. CAR T-cell peak and AUC0-28 correlated with higher ORR (P=.0224 and .0054, respectively) and increased Grade (Gr) ≥3 neurologic events (NEs; P=.0006) but not with durability of response (P=.4894). Rapid transient increases in serum analytes, including granzyme B, ferritin, IL-6, IL-10, CXCL-10, IL-15, ICAM-1, and GM-CSF, occurred early (median peak ≤7 days) and were associated with increased Gr ≥3 NEs and Gr ≥3 cytokine release syndrome (CRS; P<.05). Infusion products richer in naive-like T cells expressing CD27 and CD28 associated with increased EFS, ORR, and complete response (P<.05). In contrast, infusion products richer in differentiated T cells (CCR7-) and with lower % of CCR7+CD45RA+ T cells associated with higher postinfusion peak levels and AUC0-28 of several proinflammatory and immunomodulatory serum analytes (eg, IL-15, ferritin, IFN-γ). Increased rates of Gr ≥3 NEs were found in patients who received axi-cel richer in CCR7- T cells (above median: 30% vs below median: 10%). Similarly, a trend of higher rates of Gr ≥3 NEs and CRS were observed in patients who received axi-cel that secreted higher levels of IFN-γ in co-culture with CD19-expressing targets. Conclusions: Preinfusion axi-cel features and postinfusion PK/PD profiles in the randomized phase 3 ZUMA-7 trial were associated with safety and efficacy outcomes and supported that optimizing product composition towards a juvenile T-cell phenotype (CCR7+CD45RA+) may improve axi-cel therapeutic index. These findings could result in future trials to evaluate if preemptive interventions, including enrichment of naive T cells in the product, could improve outcomes. [SF and SV contributed equally] Citation Format: Simone Filosto, Saran Vardhanabhuti, Miguel Canales, Xavier Poiré, Lazaros J. Lekakis, Sven de Vos, Craig A. Portell, Zixing Wang, Christina To, Paul Cheng, Justin Chou, Adrian Bot, Rhine Shen, Jason R. Westin. Product attributes of axicabtagene ciloleucel (axi-cel) that associate differentially with efficacy and toxicity in second-line large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT004

Pre- and Post-Treatment Immune Contexture Correlates with Long Term Response in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel (axi-cel)

Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after 1 prior therapy as a result of ZUMA-7 (NCT. In ZUMA-1 (NCT02348216), the pivotal study which initially led to the approval of axi-cel in the 3L+ setting, the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019) and at 5 years follow up an OS rate of 42.6%, 5 years durable response and 5 years disease specific survival at 51% ( Neelapu et al. Blood 2023). T cell-related biology (Immunosign 21; Immunoscore®IC) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel ( Scholler et al., 2022). Increased density of activated PD-1+LAG-3+/−TIM-3−CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post-axi-cel. This expanded analysis characterized the impact of axi-cel treatment on the TME immune contexture and examined associations between immune cell subsets and relapse. Methods: Baseline and post-infusion tumor biopsy samples were analyzed by multiplex immunohistochemistry ( Brightplex®). Four panels were developed and applied to assess T cell infiltration (CD3 CD8 FOXP3 TIM3 PD1 LAG3 TOX), regulatory T cell subtyping (CD3 CD8 GATA3 TBET RORg BCL6), T cell activation & exhaustion (CD3 CD8 TIM3 LAG3 PD1 GZMB KI67), macrophage (CD68 CD64 CD163 CD204 CD206 PDL1) and MDSC (CD3 CD11B CD68 CD14 CD15 LOX1 S100A9) subsets. Transcriptomic analysis was performed using nCounter® Pan Cancer panel. The association between T cell subtypes and macrophages cell subset density, and probability to relapse was evaluated. T test values were based on Brightplex analysis. Results: 34 tumor biopsies (16 at baseline, 18 post-infusion) from 26 pts were analyzed including 11 relapsed (6 CR/5 PR) and 15 durable response (15 CR). The baseline and post-infusion TME comprised all major macrophages, MDSC and T cell subsets, with diverse distribution across samples analyzed. Low proinflammatory M1 macrophage density was observed at baseline and post-infusion across patients. In relapsed patients, baseline tumors were enriched with a significantly higher proportion of protumoral M2 macrophage (p<0.0001). A shift of M1-M2 polarization was observed between baseline and post-infusion (Baseline 33%M1M2 / 65%M2, post infusion 76%M1M2 / 21%M2, p<0,0001) across the cohort. In contrast, in relapsed patients, M-MDSC cell density was significantly increased post treatment (p=0.0420). In addition, Innate lymphocyte cells subtype 2 (ILC2) cell density, previously described to be positively correlated with improved overall survival in CRC ( Huang et al. 2021 Cancers), was significantly decreased in relapsed patients post infusion (p=0.019).Post-infusion, durable response was associated with a significant increase in pan-Macrophages (p=0.0360) and cytotoxic T cell subset densities: CD8 naïve T cells (p=0.016); Activated Cytotoxic T cell (GZMB+)(p=0.0203); PD1+TIM3+LAG3- cytotoxic T cell (p=0.0117); CD8 regulatory T cell (FOXP3+)(p=0.012). Regarding T helper lineage, ongoing response was associated with a significant increase of: CD4 naïve T cells (p=0.021); T helper Th2 (p=0.020); cytotoxic T lymphocyte TC2 and TC1/TC2 cell densities (respectively p=0.0052 and 0.011).Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusion: These results suggest that baseline proportions of protumor M2 macrophages may predict disease relapse after axi-cel treatment. Following infusion, relapse is also associated with spatial enrichment of M-MDSC, whereas durable response is related to increases in T cell subpopulation densities inclusive of cytotoxic T cells, T helper, TC1/TC2 ratios and innate T-subpopulations. These findings suggest that axi-cel treatment drastically impacts tumor immune mobilization, infiltration and contexture, which correlates with long term response

Product attributes of CAR T-cell therapy differentially associate with efficacy and toxicity in second-line large B-cell lymphoma (ZUMA-7)

Treatment resistance and toxicities remain a risk following chimeric antigen receptor (CAR) T-cell therapy. Herein, we report pharmacokinetics, pharmacodynamics, and product and apheresis attributes associated with outcomes among patients with relapsed/refractory large B-cell lymphoma treated with axicabtagene ciloleucel (axi-cel) in ZUMA-7. Axi-cel peak expansion associated with clinical response and toxicity, but not response durability. In apheresis material and final product, a naive T-cell phenotype (CCR7+CD45RA+) expressing CD27 and CD28 associated with improved response durability, event-free survival, progression-free survival, and a lower number of prior therapies. This phenotype was not associated with high-grade cytokine release syndrome (CRS) or neurologic events. Higher baseline and postinfusion levels of serum inflammatory markers associated with differentiated/effector products, reduced efficacy, and increased CRS and neurologic events, thus suggesting targets for intervention. These data support better outcomes with earlier CAR T-cell intervention and may improve patient care by informing on predictive biomarkers and development of next-generation products