Efficacy and safety of chimeric antigen receptor T cell therapy in relapsed/refractory diffuse large B-cell lymphoma with different HBV status: a retrospective study from a single center

BackgroundChimeric antigen receptor T cell (CAR-T) therapy is an effective salvage treatment in relapsed or refractory(r/r) diffuse large B-cell lymphoma (DLBCL), but the impact of hepatitis B virus (HBV) infection has not been studied.Methods and resultsHere, 51 patients with r/r DLBCL receiving CAR-T therapy were enrolled and analyzed at the First Affiliated Hospital of Soochow University. The overall response rate and the complete remission rate (CR) of CAR-T therapy were 74.5% and 39.2%, respectively. With a median follow-up of 21.1 months after CAR-T, the probabilities of overall survival (OS) and progression-free survival (PFS) at 36 months were 43.4% and 28.7%, respectively. These patients were divided into three cohorts including chronic HBV infection group (n=6), resolved HBV infection group (n=25) and non-HBV infection group (n=20). Bone marrow involvement was significantly higher in the HBV infection group(P<0.001), other basic characteristics before CAR-T therapy were comparable. Subgroup analysis showed that HBV infection status did not affect the efficacy of CAR-T therapy in CR rate, OS or PFS, and there was no significant difference in CAR-T related toxicities between three cohorts. Only one cirrhosis patient with chronic HBV infection experienced HBV reactivation.ConclusionsCAR-T therapy was effective and can be used safely in r/r DLBCL with HBV infection under proper monitoring and antiviral prophylaxis

Body mass index-associated responses to an ABVD-like regimen in newly-diagnosed patients with Hodgkin lymphoma

Background: The role of body mass index (BMI) in the treatment outcomes of lymphoma patients is controversial. While investigating the efficacy of ABVD-like regimen in Hodgkin lymphoma (HL) patients, we observed that obese patients had poor responses. To better understand this clinical phenomenon, we evaluated the effect of BMI on responses to ABVD-like chemotherapy in HL patients.Methods: This retrospective cohort study evaluated the clinical outcomes of all 67 patients with confirmed HL who were treated at the First Affiliated Hospital of Soochow University from November 2016 to March 2023 with an ABVD-like regimen as first-line chemotherapy. Baseline patient characteristics and clinical outcomes were compared across different BMI categories. The primary end-point was the overall response rate defined as the proportion of the HL patients who achieved complete response or partial response. The additional end-points included progression-free survival and overall survival.Results: The median age of the HL patients was 31 years old. Of the patients, 10.4% were obese, and 17.9% patients were overweight. Interim and end-term response evaluations revealed overall response rates of 98.5% and 83.6%, respectively. The proportion of patients with potential poor prognostic factors (IPS risk factors) did not differ significantly in the responders versus non-responders. However, non-responders had a higher average BMI when compared with responders (p = 0.002). Poor overall response rates in higher BMI patients indeed manifested with shorter progression free survival (p = 0.013). The minimum relative dose of the ABVD-like regimen in the overweight and obese groups was significantly lower than in the normal weight group (p < 0.001).Conclusion: Our analyses show that >80% of newly-diagnosed HL patients responded to the ABVD-like regimen. We find that being obese or overweight at the time of diagnosis correlated with a poorer overall response rate and that BMI was an independent risk factor in HL patients treated with the ABVD-like regimen. Lower doses of ABVD-like regimen contributed to the discrepant findings of responses in the high BMI groups. These findings indicate that newly-diagnosed, obese HL patients receiving an ABVD-like regimen require personalized treatment

Circulating tumor DNA determining hyperprogressive disease after CAR-T therapy alarms in DLBCL: a case report and literature review

Chimeric antigen receptor T-cell therapy (CAR-T) has been widely applied in the clinical practice of relapse/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) due to its promising effects. Hyperprogressive disease (HPD) has gained attention for rapid tumor progression and has become a therapeutic and prognostic challenge. Here, we present a patient who had suffered from several recurrences previously and controlled well with a very small tumor lesion left was infused with CD19/CD22 bispecific CAR-T, with no immune effector cell-associated neurotoxicity syndrome, or cytokine release syndrome observed. However, rapid deterioration, subsequent imaging examination, circulating tumor DNA, and serum biomarkers detection identified HPD. The patient did not respond to salvage treatment and died 40 days after infusion. To our knowledge, only one case of HPD in DLBCL after CAR-T therapy has been reported. This fatal case alarmed the risk of HPD and the ctDNA profile monitoring we used was performed as a non-invasive method to diagnose HPD, providing far-reaching practical instruction for CAR-T therapy

ABCG2 is a Direct Transcriptional Target of Hedgehog Signaling and Involved in Stroma-Induced Drug Tolerance in Diffuse Large B-Cell Lymphoma

Successful treatment of diffuse large B-cell lymphoma (DLBCL) is frequently hindered by development of resistance to conventional chemotherapy resulting in disease relapse and high mortality. High expression of anti-apoptotic and/or drug transporter proteins induced by oncogenic signaling pathways has been implicated in the development of chemoresistance in cancer. Previously, our studies showed high expression of ATP-binding cassette drug transporter ABCG2 in DLBCL correlated inversely with disease-free and failure-free survival. In this study, we have implicated activated hedgehog (Hh) signaling pathway as a key factor behind high ABCG2 expression in DLBCL through direct upregulation of ABCG2 gene transcription. We have identified a single binding site for GLI transcription factors in the ABCG2 promoter and established its functionality using luciferase reporter, site-directed mutagenesis and chromatin-immunoprecipitation assays. Furthermore, in DLBCL tumor samples, significantly high ABCG2 and GLI1 levels were found in DLBCL tumors with lymph node involvement in comparison to DLBCL tumor cells collected from pleural and/or peritoneal effusions. This suggests a role for the stromal microenvironment in maintaining high levels of ABCG2 and GLI1. Accordingly, in vitro co-culture of DLBCL cells with HS-5 stromal cells increased ABCG2 mRNA and protein levels by paracrine activation of Hh signaling. In addition to ABCG2, co-culture of DLBCL cells with HS-5 cells also resulted in increase expression of the antiapoptotic proteins BCL2, BCL-xL and BCL2A1 and in induced chemotolerance to doxorubicin and methotrexate, drugs routinely used for the treatment of DLBCL. Similarly, activation of Hh signaling in DLBCL cell lines with recombinant Shh N-terminal peptide resulted in increased expression of BCL2 and ABCG2 associated with increased chemotolerance. Finally, functional inhibition of ABCG2 drug efflux activity with fumitremorgin (FTC) or inhibition of Hh signaling with cyclopamine-KAAD abrogated the stroma-induced chemotolerance suggesting that targeting ABCG2 and Hh signaling may have therapeutic value in overcoming chemoresistance in DLBCL

Smoothened Stabilizes and Protects TRAF6 from Proteosomal Degradation: A Novel Non-Canonical Role of Smoothened with Implications in Lymphoma Biology

Abstract Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a crucial role in many biological processes and its activity is relevant in the biology of multiple cancers including diffuse large B cell lymphoma (DLBCL). Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 levels and/or enhance TRAF6 function remain largely unclear. Previously, we found that activation of smoothened (SMO) with recombinant Hedgehog (Hh) ligand increased the binding between SMO with TRAF6, as well as TRAF6 protein levels (Blood 2013; 121:4718-28). In addition, transient overexpression of SMO resulted in increased K63-Ub of both TRAF6 and NEMO indicating stabilization of these proteins resulting in NF-kB activation. This is relevant, as more recently we found that TRAF6 amplifies pAKT signaling in DLBCL and that TRAF6 is the dominant E3 ligase for the K63-Ub of AKT in DLBCL. Moreover, TRAF6 recruitment to the cell membrane, and stabilization of its ubiquitination profile are facilitated by SMO. SMO is a member of the Frizzled-class G-protein-coupled receptor (GPCRs) and is traditionally known for its role as signal transducer in canonical Hedgehog (Hh) signaling. These observations prompted us to investigate whether the ability of SMO to increase TRAF6 levels is limited to ligand induced signaling, whether it contributes to chemoresistance in DLBCL cells, and whether SMO directly participates in controlling TRAF6 levels. To confirm the regulatory role of SMO in the TRAF6/AKT axis in DLBCL cells (HBL1 and HT) and further outline the nature of the underlying regulation, we measured the impact of activation of the Hh pathway with recombinant Shh ligand on TRAF6 levels, with and without SMO knockdown or recombinant SMO overexpression. Canonical Hh signaling results in the activation of the GLI1 transcription factor and the subsequent elevation of GLI1 mRNA levels is an established indicator of activation of the Hh pathway. However, neither SMO activation nor the knockdown of GLI1 had a significant impact on TRAF6 mRNA levels. These findings indicate that TRAF6 is not transcriptionally regulated by SMO signaling through GLI1 (canonical Hh signaling). In contrast, overexpression of SMO or siRNA knockdown of SMO resulted in an increase or decrease of TRAF6 protein levels, respectively. Consistent with the decrease of AKT activation (pAKT T308 and S473) after TRAF6 knockdown, the increase in TRAF6 levels that follows SMO overexpression resulted in an increase in the levels of AKT phosphorylation. Altogether, these observations suggest a post-translational regulation of TRAF6 by SMO. Indeed, stable knockdown of SMO dramatically reduces the half-life of TRAF6 in both HBL1 and HT cells in the presence of cyclohexamide. Furthermore, overexpression of SMO increases K63-Ub of both TRAF6 and AKT. In contrast, the SMO induced decrease in K48-Ub occurred only for TRAF6 but not for AKT. These data link the SMO-stimulated activation of TRAF6 to the enhancement of AKT signaling and protection of TRAF6 from proteasomal degradation. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes TRAF6 and protects TRAF6 from proteosomal degradation, an effect mediated by ubiquitin-specific protease-8 (USP8). Importantly, this functional link between SMO and TRAF6 is reflected in DLBCL patient samples where high expression of both molecules correlates with poor prognosis. Resistance to DXR is a serious challenge in the treatment of DLBCL, and activated AKT is known to contribute to DXR resistance in multiple cancers including DLBCL. We evaluated whether SMO and TRAF6 support resistance to DXR in DLBCL cell lines. We exposed HT and HBL1 cells as well as their counterparts with stable knockdown of TRAF6 or SMO to DXR for 96hrs. Cell viability after exposure to DXR was determined by an Annexin V and PI staining assay. Silencing SMO or TRAF6 dramatically decreased cell survival after treatment with DXR. In summary, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels in DLBCL cell lines of germinal (GC) and non-GC subtypes, and that the SMO/TRAF6 axis contributes to DXR resistance in DLBCL. Our study reveals a novel and potential central cell survival signaling mechanism in which SMO stabilizes and protects TRAF6 from proteosomal degradation. Disclosures Lossos: Affimed: Research Funding

Smoothened stabilizes and protects TRAF6 from degradation: A novel non-canonical role of smoothened with implications in lymphoma biology

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a role in many biological processes and its activity is relevant in diffuse large B cell lymphoma (DLBCL) biology. Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 and/or enhance TRAF6 function remain largely unclear. We found that TRAF6 amplifies pAKT signaling in DLBCL. Moreover, TRAF6 activation and stabilization of its ubiquitination profile are facilitated by smoothened (SMO), signal transducer of canonical Hedgehog signaling. Here, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels, and that the SMO/TRAF6 axis contributes to doxorubicin resistance in DLBCL. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes and protects TRAF6 from degradation, an effect mediated by ubiquitin-specific protease-8. Moreover, this functional link between SMO and TRAF6 is reflected in DLBCL patients where high expression of both molecules correlates with poor prognosis. In summary, our study reveals a novel cell survival mechanism in which SMO stabilizes and protects TRAF6 from degradation. The axis SMO/TRAF6/AKT is highly relevant in the biology of DLBCL and is involved in doxorubicin resistance. •TRAF6 amplifies pAKT signaling in DLBCL.•SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels.•SMO stabilizes and protects TRAF6 from proteosomal degradation.•High expression of SMO and TRAF6 correlates with poor prognosis in patients with diffuse large B cell lymphoma.•SMO/TRAF6/AKT axis contributes to doxorubicin resistance in DLBCL

Defining Causative Factors Contributing in the Activation of Hedgehog Signaling in Diffuse Large B-Cell Lymphoma

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-KB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling