Abstract Topoisomerase 1 (TOP1) is a ubiquitous enzyme that primarily regulates the topology of DNA by governing the relaxation of DNA supercoiling. When TOP1 is inhibited by camptothecin and its analogs, re-ligation of TOP1 cleavage complexes (TOP1cc) is prevented, leading to the accumulation of TOP1cc-associated DNA damage, inhibition of cellular proliferation, and eventual cell death. Antibody drug conjugates (ADCs) are a class of cancer therapeutics in which a potent cytotoxic payload is linked to a tumor-targeting antibody. ADCs allow direct delivery of a drug to tumor cells, thus enhancing tumor cell-specific killing. Several TOP1 inhibitor-ADCs (TOP1i-ADCs) have been approved, and others are under clinical investigation, showing efficacy in solid tumors. Beyond direct cytotoxicity, recent evidence suggests that TOP1i and TOP1i-ADCs trigger immunogenic changes in cancer cells, fostering immune activation. In studies using colorectal (CRC) cell lines treated with adizutecan, a proprietary AbbVie TOP1 inhibitor, we observed upregulation of gene pathways involved in the Type I Interferon response. Notably, adizutecan increased surface expression of immunogenic markers including MHC-I, MHC-II, CALR, and PD-L1 in CRC lines. To evaluate TOP1i-ADC-induced immune activation within a native tumor microenvironment, organotypic tumor slices from fresh human metastatic colorectal carcinoma were utilized for this study. We treated these fresh human metastatic colorectal carcinoma tissue slices with TOP1i-ADC and evaluated changes in both the tumor and immune cells by flow cytometry and spatial transcriptomics. TOP1i-ADC treatment led to reduced tumor cell proliferation and viability, and consistent activation of dendritic cells as well as CD4 T cells. CD8 T cell activation was observed in 3 out of 5 explant samples. The flow cytometry data was further supported by the secretome profile, which shows elevated levels of pro-inflammatory chemokines and cytokines such as CCL3, CXCL10, G-CSF and granzyme B (GZMB). Spatial transcriptomics analysis revealed TOP1i-ADC treatment reduced DNA replication and cell cycle gene signatures consistent with TOP1i payload release within the tumor explant. Consistent with the flow cytometry data, immune cell responses displayed dendritic cell maturation in both proximal and distal regions to the tumor cells. CD8 T cell activation, evidenced by higher granzyme B (GZMB) expression, was especially prominent in distal areas consistent with spatially distinct regional activation. Collectively, these findings support that TOP1i-ADC therapy drives potent immune activation and immunogenicity, complementing its direct antitumor effects and highlighting its promise for enhancing cancer treatment efficacy. Citation Format: Irene I. Lee, Durga B. Dandamudi, Yoshiko Hashikawa, Marion L. Refici, Michelle Belmont, Iraz Aydin, Rhyannon Spangler, Steven Chirieleison, David Sharon, PK Epling-Burnette, Athan Vasilopoulos, Tamar Uziel, Jack Chen, Lloyd Lam. TOP1i-ADC demonstrates immune-stimulating activity in colorectal cancer explants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 663.

Several new therapies have been approved for the treatment of B-cell NHL in the relapsed/refractory setting. CD3xCD20 bispecific antibodies and chimeric antigen receptor T-cell (CAR T) therapy are powerful immunotherapies that activate T-cells to kill CD20- or CD19-expressing malignant cells. Approved CAR T therapies rely on structural engineering and adoptive transfer of autologous T-cells following lymphodepletion, while CD3xCD20 bispecifics are engineered antibodies that recruit and redirect the activity of endogenous T-cells against tumor. The aim of this study is to describe the phenotypic composition of endogenous T-cells and CAR T post-CAR T therapy and assess function of these cells ex vivo in response to CD3xCD20 bispecifics. Clinical response data, endogenous T-cell characteristics, and ex vivo activity of viably frozen peripheral blood mononuclear cells (PBMCs) in the presence of CD3xCD20 bispecific antibodies were analyzed from 22 patients (diffuse large B-cell lymphoma [DLBCL], n=19; follicular lymphoma [FL], n=3) from the Montpellier University Hospital after written informed consent in accordance with the Declaration of Helsinki and institutional research board approval. Patients received axicabtagene ciloleucel (Yescarta, Gilead Sciences; n=17) or tisagenlecleucel (Kymriah, Novartis; n=5), both CD19-targeted CAR T. In this study, all patients received CAR T therapy after ≥2 prior lines of therapy and CAR T was detected using flow cytometry from the Montpellier University Hospital. In ex vivo experiments, both glofitamab and epcoritamab were tested for methods development and feasibility. Epcoritamab was then used to assess functionality of the full cohort of PBMC samples collected five days before (D -5; at the time of apheresis), and at 3 to 6 months (M3, M6) after CAR T therapy. Phenotypic assessments were performed throughout therapy by flow cytometry including D -5, after the standard conditioning regimen containing cyclophosphamide and fludarabine 2 to 3 days based on the CAR T therapy, and up to 300+ days post-CAR T therapy. In this CAR T-treated cohort, complete responses were observed in 81.8% (n=18/22) with durability of complete response from 7 months to 4 years and included patients with DLBCL and FL. Four patients relapsed within 3 to 6 months with only 1 of the early relapsed patients showing no CAR T expansion. Endogenous T-cells and CAR T were independently assessed and were found to expand with different kinetics, peak at 7 to 10 days for CAR T, and later (~10–300+ days) for endogenous T-cells. In paired analysis pre- to post-CAR T infusion, CD8 T-cells expanded and CD4 T-cells decreased in both responders and nonresponders. In patients with complete response, a significant expansion of endogenous CD8+ effector memory T-cells (P<.01) was observed without increase in LAG3, PD-1, or TIM3. A similar trend was observed in patients with progressive disease. Endogenous T-cells in PBMC samples collected pre- (D -5) and post- (M3, M6) CAR T therapy showed similar ex vivo cytotoxic activity and exhibited similar induction of the activation antigen CD25 in response to either epcoritamab, glofitamab, or anti-CD3/CD28 in both CAR T responders and nonresponders. Endogenous T-cells post-CAR T therapy in DLBCL and FL were functionally responsive to epcoritamab. Phenotypic changes post-CAR T therapy were consistent with T-cell reconstitution following lymphopenia post-cytoreduction where the T-cells proliferate in response to homeostatic cytokines that favor CD8+ T-cells with a specific effector/effector memory phenotype (Williams et al. Semin Immunol, 2007). The phenotypic changes were independent of the kinetics and response to CAR T therapy, suggesting that bispecific engagement can activate and redirect T-cells even in patients who failed CAR T therapy. This data supports the benefit of epcoritamab post-CAR T therapy, as evidenced by the high complete response rate observed in epcoritamab-treated CAR T-exposed patients (Thieblemont C, et al. Leukemia 2024).

Introduction: Etentamig is a differentiated, 2nd generation, BCMA x CD3 bispecific T-cell engager composed of a high avidity bivalent BCMA-binding domain, a low-affinity CD3-binding domain designed to reduce cytokine release syndrome, and a silenced Fc tail for extended half-life, enabling convenient dosing. Recent findings from a Phase 1b, open-label, dose-escalation and expansion trial (NCT05259839) showed that etentamig in combination with daratumumab, a CD38 monoclonal antibody, and dexamethasone demonstrated promising activity in heavily pre-treated relapsed/refractory multiple myeloma (RRMM) (Blood 2024;144(Supplement 1):496). The current study aims to further elucidate the mechanisms associated with daratumumab enhancement of etentamig activity through ex vivo studies. Methods: Peripheral blood mononuclear cells (PBMCs) and T cells, isolated from either healthy donors or myeloma patients, were co-cultured with MM cell lines (RPMI-8226, NCI-H929, LP-1, and U266B1). The co-cultures were treated with either etentamig alone or in combination with daratumumab for 4 days prior to assessment of tumor cell lysis and T-cell activation, immune modulation, and cytokine secretion through flow cytometric analysis and multiplex cytokine quantification (Luminex FLEXMAP 3D technology), respectively. Additionally, immunosuppressive T cell populations were quantified via multiparameter flow cytometric data from samples derived from newly diagnosed, daratumumab-naïve, and post daratumumab therapy MM patients. Results: Etentamig and daratumumab effectively induced the lysis of MM cell lines in co-culture with healthy donor and MM PBMCs. The amount of tumor cell lysis was significantly enhanced by the combination compared with either therapeutic molecule alone. Interestingly, etentamig-mediated MM lysis was enhanced with PBMCs from RRMM patients who had been exposed to daratumumab compared with daratumumab-naïve RRMM patients. In co-culture assays, etentamig in combination with daratumumab had reduced levels of forkhead box P3 (FOXP3)+ regulatory T cells (Treg) compared with etentamig treatment alone, as well as reduced levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that plays an immunosuppressive role in the bone marrow microenvironment. Detailed immunophenotypic analysis through multiparameter flow cytometry highlighted the impact of daratumumab treatment on several Treg subsets characterized by CD38 expression. CD38+ Treg cells were previously shown to be more suppressive in vitro than CD38-negative Tregs and expressed additional markers indicative of their heightened immunosuppressive activity, such as inducible T-cell costimulator, HLA-DR, CD39, and Ki67. In parallel, etentamig in combination with daratumumab resulted in enriched programmed cell death protein 1 (PD1)-negative CD8+ T cells, with minimal impact on etentamig-induced T cell activation (CD8+CD25+) and proliferation markers (CD8+Ki67+). Furthermore, daratumumab treatment reduced levels of CD8+ T cells expressing PD1 and thymocyte selection-associated HMG-box (TOX), a gene that plays a crucial role in the induction of T cell dysfunction. Conclusions: We show that daratumumab enhances anti-tumor activity of etentamig by i) augmenting direct anti-tumor activity, ii) depleting immune regulatory and dysfunctional T cell populations, and iii) modulating the immunosuppressive bone marrow microenvironment (eg, IDO reduction). Collectively, our findings support the continued investigation of etentamig in combination with daratumumab in patients with MM.

Telisotuzumab Vedotin (Teliso-V), a first-in-class c-Met-MMAE antibody-drug conjugate (ADC), achieved FDA breakthrough therapy designation in c-Met overexpressing (OE), 2nd Line+ EGFR wild-type, non-squamous, non-small cell lung cancer (NSCLC) patients. In addition, in a phase 1/1b trial, Teliso-V demonstrated 50% response in combination with the EGFR inhibitor, osimertinib, in EGFR mutant (EGFR-MT) NSCLC patients who had progressed on osimertinib. Here, we present mechanistic insights into the combinatorial effects of Teliso-V with osimertinib in this patient population. EGFR-MT NSCLC cell lines were exposed to osimertinib and subjected to RNA expression, mutation, EGFR-interactome, and ADC intracellular localization and endolysosomal (EL) trafficking analysis. While no mutation gains or losses were detected, chronic and acute treatment with osimertinib enhanced Teliso-V sensitivity in EGFR-MT NSCLC cells. In addition to enhanced Teliso-V cytotoxicity, we observed enhanced accumulation of Teliso-V in a low pH intracellular compartment, consistent with increased endolysosome (EL) trafficking. Chronic osimertinib exposure induced gene expression changes related to increased lysosomal membranes, lytic vacuole membranes, and endoplasmic reticulum as well as reduced EGFR signaling. In addition to osimertinib, other EGFR inhibitors including erlotinib, gefitnib, or afatinib also enhanced Teliso-V EL trafficking which is consistent with the effect of EGFR-MT signaling in suppressing c-Met ADC EL trafficking. Cells harboring EGFR-MT are known to form large multimeric protein complexes to regulate EGFR trafficking and cell survival. EGFR-MT immunoprecipitation, followed by mass spectrometry analysis identified 84 proteins associated with an osimertinib-sensitive complex including c-Met, and caveolin-1 (CAV-1), a structural protein involved in signaling and trafficking. Confocal microscopy imaging showed that osimertinib treatment altered the subcellular localization of these proteins and their interaction with CAV-1. CAV-1 knockdown disrupted the multimeric protein complex, suppressed EGFR and c-Met activation, reduced downstream AKT/ERK signaling, and enhanced Teliso-V-associated EL trafficking, phenocopying osimertinib effect. Chronic and acute treatment with osimertinib displaces EGFR/c-Met/CAV-1 multimeric signaling complexes leading to improved lysosomal processing of c-Met-targeted ADCs and enhanced tumor cytotoxicity. This study provides mechanistic insights into the combination of Teliso-V and osimertnib in 2L+ EGFRMT NSCLC patients in the clinic. Izhar Batth, Ronald Pandoy, Lisa Roberts-Rapp, Athan Vasilopoulos, Peter Ansell, Vincent Blot, Gregory K. Potts, Janice Y. Lee, Hua Tang, Jon D. Williams, Tamar Uziel, PK Epling-Burnette. Elucidating the mechanism of clinical Teliso-v (cMet-MMAE ADC) and osimertinib combination: A CAV1 and EGFR multi-protein complex controls endolysosomal trafficking of Teliso-V [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4713.

Epcoritamab (epcor) is a subcutaneously (SC) administered bispecific T-cell engager (CD3-BsAb TCE) approved for the treatment of relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) after ≥2 lines of therapy. Functionality of the immune compartment may impact rates and duration of response. Here, we report the clinical outcome of epcor monotherapy administered in the outpatient setting in DLBCL and show changes in clonally reactive T-cells with improved functional states linked to long-term response. Patients in the EPCORE NHL6 phase 2 trial (NCT05451810) received epcor in 28-day cycles (C): two C1 step-up doses (0.16mg, 0.8mg) then full dose at C1D15 and thereafter (48mg); C1-3, QW; C4-9, Q2W; C≥10, Q4W. Responses were investigator-assessed per Lugano criteria. Multi-parameter flow cytometry, ex vivo cytotoxicity, cytokine and T-cell activation induced by epcor were tested in the presence of a CD20-expressing tumor cell line (Jeko-1) using peripheral blood mononuclear cells (PBMCs) collected at baseline and after 2, 4, 5, and 7 cycles of epcor treatment from n=43 patients. PBMCs from samples collected pre- and on-treatment in a subset of patients that achieved response (complete response [CR]/partial response [PR]; n=7) were evaluated using Single-cell Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) and single-cell T-cell receptor (TCR) repertoire analysis. At the 15 January 2025 cutoff, 92 patients received ≥1 epcor dose (response and adverse events reported previously [Vaidya R, et al. SOHO 2025]). The overall response rate (ORR) for the 92 patients was 62.0% (95% CI: 51.2%, 71.9%) and the CR rate (CRR) was 42.4% (95% CI: 32.1%, 53.1%), which was similar in the subset of patients (n=43) with immune profiling (ORR, 62.8% [95% CI: 46.7%, 77.0%]; CRR, 44.2% [95% CI: 29.1%, 60.1%]). Ex vivo functional studies showed significant improvement in cytotoxic activity in a time-dependent and T-cell-dependent manner with higher proportions ofCD3 T-cells expressing activation and proliferation markers (CD25, P=.008; CD69, P=.041; Ki67, P=.001) in longitudinal samples from patients who achieved CR (n=13) vs patients with progressive disease (PD; n=10). UMAP analysis of flow cytometry data identified an accumulation of Tbethigh effector memory CD8 T-cells expressing granzyme B (P=.060) and a significant decrease (P=.036) in CD8+ T-cells with a senescence phenotype (CD28low, CD27low,CD57+ TEMRA cells positive for β-galactosidase) in CR patients. These changes were associated with improved ex vivo T-cell cytotoxic function in patients achieving a CR. Sorting experiments demonstrated that the ex vivo cytotoxic effects of epcor is primarily mediated by the non-senescent (CD8+CD57-) T-cell population. Preliminary CITEseq analysis showed that circulating CD8 T-cells, including CD8 effector memory (TEM), terminally differentiated TEM cells that express CD45RA (TEMRA), and effector cells, were activated and maintained a low exhaustion status during epcor treatment. Single-cell TCR repertoire analysis showed overlap of TCR sequences between baseline and on-treatment samples and examples of TCR clone expansion with epcor treatment among responders. These findings highlight the immunologic impact of epcor treatment, marked by a progressive increase in functionally active CD8+ T cells with examples of increased TCR clonality and a reduction in senescent T-cell populations, notably without evidence of emerging dysfunction. These data provide insight into the mechanism of action of epcor and support its continued development as a transformative therapy in B-cell lymphoma.

Abstract Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of a c-Met (MET protein) antibody coupled to the microtubule inhibitor monomethyl auristatin E (MMAE). Teliso-V was granted FDA Breakthrough Therapy Designation for advanced/metastatic EGFR WT, NSQ NSCLC with high c-MET overexpression. For MMAE drug delivery, Teliso-V must first undergo receptor internalization and endolysosomal (EL) trafficking. Hepatocyte growth factor (HGF)-dependent MET EL trafficking and degradation depend on the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL). METex14 skipping and CBL-binding site mutations, which delete the binding site for CBL, occur in ~ 3% of NSCLC. The role of CBL in Teliso-V-MMAE delivery and activity was assessed in this work. Methods: WT-human c-Met, c-Met-Y1003F/N (CBL binding site mutations in MET), and c-Met-ex14∆ (47 aa deletion to mimic ex14 skipping) or CBL-L620f, G807*, and W802* (CBL inactivating mutants) were expressed in NIH3T3, Ba/F3 cells, or A549 tumor cells using retroviral transduction. Genetically modified cells were then used to define the role of CBL in c-Met surface expression, intracellular signaling, EL trafficking (using pHrodo Red-labeled Teliso-V), and Teliso-V sensitivity in 2D/3D cell cultures. Results: c-Met expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. While minimal changes in cell surface c-Met levels were observed, CBL binding mutants had increased phosphorylated c-Met in the absence of HGF, and ex14∆-MET and CBL binding site mutants showed increased sensitivity to small molecule c-Met inhibitors. Compared to WT c-Met, ex14∆ and CBL-binding mutants showed similar EL trafficking and no change in sensitivity to Teliso-V. Co-treatment with a pan-CBL inhibitor also failed to alter Teliso-V EL trafficking and anti-tumor activity. Like most ADCs, EL trafficking was dependent on a dynamin-mediated process with reduced EL trafficking by co-treatment with Dyngo4a. To further understand the role of CBL in Teliso-V sensitivity, we expressed CBL inactivating mutations in A549 tumor cells. While the L602F and W802* mutants increased spheroid size, as well as activated p-MET, there was no change in sensitivity to Teliso-V. Conclusions: Loss of CBL-binding sites on c-Met via ex14 deletion or Y1003F/N leads to increased MET-targeted TKI sensitivity and increased phospho-c-Met levels but does not impact Teliso-V sensitivity or EL trafficking. Our results indicate that Teliso-V traffics to the EL via a dynamin-mediated process. Therefore, Teliso-V delivers MMAE through a robust ADC-mediated EL-dependent mechanism that is independent of CBL, suggesting that high c-Met expression in Ex14skipping or CBL binding site mutant NSCLC patients should be further investigated for response to Teliso-V. Citation Format: Izhar Batth, Ronald Pandoy, Shamim Khaja, Dolonchampa Maji, Tamar Uziel, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. Telisotuzumab vedotin (Teliso-V)-mediated c-Met internalization, endolysosomal trafficking, and potent cytotoxic activity are unaffected by MET exon-14 skipping mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2116.

Background: Venetoclax (ven) in combination with hypomethylating agents or low dose cytarabine leads to rapid and durable remission in patients (pts) with acute myeloid leukemia (AML) unfit for intensive chemotherapy (IC), however, pts with TP53 mutations (TP53mut) exhibit adverse prognosis. Here, we identify dysregulation of the integrated stress response (ISR) pathway in pts with TP53mut, specifically through the actions of DAP3 binding cell death enhancer 1 (DELE1) and its activating protease OMA1. Methods: RNA sequencing was performed on pre-treatment bone marrow-derived mononuclear cells (BMMCs) from pts with AML ineligible for IC across four clinical trials (NCT02993523, NCT02203773, NCT03069352, and NCT02287233). CRISPR-Cas9 editing of TP53-intact AML cell lines was used to generate TP53-/-, TP53mut, DELE1-/-, OMA1-/- and TP53/DELE1 double deficient cell lines. Quantification of BCL2 and Myeloid cell leukemia 1 (MCL1) complexes with BCL2 interacting mediator (BIM) was performed using chemiluminescence assays. A drug screen to assess viability and gene expression was performed in cells treated with ven in combination with 63 drugs. Results: In total 401 pts were included in the analyses, of which 17% harbored TP53mut and 83% were TP53wt. Analysis of genes differentially expressed (DE) between pts with TP53mut and TP53wt AML revealed 19 DE genes shared across all trials. DELE1, an ISR adaptor, was associated with genes mapping to the ISR-related eukaryotic initiation factor-α (eIF2α) in TP53mut AML BMMCs. Deletion of DELE1 or OMA1 in AML cell lines blocked eIF2α activation and induction of the transcription factor ATF4, a critical ISR effector, in response to the mitochondrial stressor FCCP, ven, and azacitidine, and resulted in ven resistance similar to that of TP53 deficient cells. All modified AML cell lines exhibited a concomitant decrease in the pro-apoptotic regulator PMAIP1, encoding NOXA, and a 14-18-fold increase in MCL1-BIM complexes following ven treatment, as well as increased MCL1 expression, compared to parental lines. Further screening of these cell lines for ven sensitizing activity revealed the BH3 mimetic S63845, which targets MCL1, as the top hit, suggesting that combined BH3 mimetics may overcome ven resistance during ISR pathway defects. As DELE1 is located on chromosome 5 (CH); the frequent loss of CH 5q among TP53mut AML may contribute to low DELE1 expression. Conclusions: These data suggest that defective ISR signaling may be a factor in TP53mut AML treatment outcome and point to DELE1 dysregulation as a driver of ISR inactivation in pts with TP53mut AML. The ISR induces the expression of NOXA, which displaces MCL1 from BIM and lowers the apoptotic threshold. Our data identify p53, DELE1, and OMA1 as regulators of NOXA expression post ven treatment and indicate that co-targeting MCL1 in pts with TP53mut AML may be beneficial to overcome ven resistance. Citation Format: David Sharon, Paul Jung, Yan Sun, Weiguo Feng, Ziping Yang, Valerie Robinson, Diya Mitra, Wei Liu, Pingping Zheng, Tamar Uziel, Lloyd Lam, Mark D. Minden, Jeremy Ross, Wellington Mendes, Jalaja Potluri, Andrew H. Wei, Marina Konopleva, Monique Dail, Brenda Chyla, PK Epling-Burnette. DELE1 loss and dysfunctional integrated stress signaling in TP53 mutated AML is a novel pathway for venetoclax resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2530.

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

The classic myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are driven by aberrant activation of the JAK2 kinase, primarily by mutations in 3 different genes, JAK2, MPL, and CALR. JAK2 inhibitor therapy is generally effective at reducing MPN patient constitutional symptoms and thus can improve quality of life but has minimal effect on reducing mutant driver allele burden and does not readily alter the course of disease, reflecting the ability of MPN-driving cells to persist during therapy. Thus, the ability of current anti-JAK2 therapies to significantly modify underlying disease biology is limited. Long-term studies of the JAK2 inhibitor ruxolitinib in myelofibrosis patients have, however, identified survival benefits, yet this may be due to anti-inflammatory and spleen reducing impacts. Ultimately, the inability of ruxolitinib and other approved JAK2 inhibitors to effectively antagonize allele burden indicates alternative therapeutic approaches are needed to overcome the persistent survival of MPN-driving cells during JAK2 inhibitor therapy. Efforts to identify mechanisms that can thwart the effects of JAK2 inhibition in MPN cells have highlighted activation of the RAS-ERK pathway, including mechanisms of RAS-ERK signaling that are independent of JAK2. ERK or MEK inhibition have been shown to enhance the activity of ruxolitinib in MPN mouse models, but monotherapeutic inhibition of these kinases displayed limited efficacy. The SHP2 (Src homology region 2 domain-containing phosphatase-2) tyrosine phosphatase mediates RAS activation and subsequent MEK/ERK signaling downstream of cytokine and growth factor receptors. SHP2 plays a role in oncogenic signaling and in resistance to kinase targeted therapies, exemplified in both solid tumor and leukemia models. Given the evidence that activation of RAS-ERK signaling can antagonize the effects of JAK2 inhibition and the fact that SHP2 mediates RAS activation via multiple mechanisms downstream of numerous growth factor and cytokine receptors, we hypothesized that SHP2 may contribute to the survival of MPN cells during JAK2 inhibitor treatment and may provide a therapeutic target to improve current anti-JAK2 therapies. Using preclinical MPN models, we aimed to block RAS activation by inhibiting SHP2 to assess the potential SHP2 inhibition may have as a monotherapy or in combination with JAK2 inhibition. We found that the SHP2 inhibitors RMC-4550 and SHP099 enhanced growth inhibition of MPN model cell lines (e.g., SET2 and UKE1) in combination with ruxolitinib, effectively preventing ruxolitinib persistent growth. Concomitant treatment with both inhibitors led to enhanced inhibition of RAS-GTP levels and ERK activation and increased apoptosis compared to single agents. SHP2 inhibition antagonized the neoplastic growth of primary hematopoietic progenitor cells from MPN patients and synergized with ruxolitinib in all patient samples assessed. In an MPN mouse model driven by MPL-W515L, RMC-4550 antagonized MPN phenotypes, including leukocytosis, hepatosplenomegaly, and allele burden. RMC-4550 treatment diminished levels of a subset of pro-inflammatory cytokines, including IL-1β, CXCL9, and TNFα, among others. The combination of RMC-4550 with ruxolitinib not only suppressed advancing leukocytosis but led to white blood cell levels lower than at treatment initiation (Panel A) and suppressed thrombocytosis compared to ruxolitinib alone. The combination also led to enhanced inhibition of hepatomegaly, extramedullary hyperplasia, megakaryocyte hyperplasia, and allele burden, and a trend toward improved fibrosis. The SHP2 inhibitor RMC-4550 enhanced the survival of mice (following termination of treatment analyses) as both a monotherapy (p < 0.0001 compared to vehicle) and in combination with ruxolitinib (p < 0.01 compared to ruxolitinib monotherapy). Importantly, the combination of SHP2 inhibition using RMC-4550 with JAK2 inhibition using ruxolitinib for 4 weeks in wildtype mice was well tolerated with respect to hematologic parameters and exemplified by no effect on body weight (Panel B). Given SHP2 inhibitors are already undergoing clinical evaluation in patients with solid tumors, our findings suggest that SHP2 is a therapeutic target with potential to be rapidly translated to clinical assessment for MPN patients.

Background: Telisotuzumab vedotin (Teliso-V) is an antibody-drug conjugate (ADC) composed of the c-MET antibody ABT-700 and the microtubule inhibitor monomethyl auristatin E (MMAE). Patients with EGFR wildtype (WT) nonsquamous non-small cell lung cancer with high c-MET expression had an overall response rate of 54% with Teliso-V. In addition to overexpression and amplification, genetic alterations in c-MET, such as exon 14 deletion (ex14del) and missense mutations in the tyrosine kinase domain (TKD), contribute to its constitutive activation. The efficacy of Teliso-V in context of these genomic backgrounds is currently unknown. Methods: Recombinant retroviruses were used to express WT-c-MET, c-MET-TKD (D1228H/V/N and Y1230C/H mutations), c-MET-Y1003F/N (CBL E3 ubiquitin ligase binding site mutation), and c-MET-ex14del (47 aa juxtamembrane deletion) mutants in NIH3T3 and Ba/F3 cells. c-MET surface expression, intracellular signaling, ADC internalization and endolysosomal (EL) trafficking (using pHrodo Red-labeled Teliso-V), intracellular MMAE by LC/MS, and Teliso-V sensitivity were studied in 2D/3D cell cultures. Full-length c-MET protein structures were created with the ColabFold program and molecular dynamics simulation software (with/without artificial lipid bilayer) was used to predict the mechanistic basis for differential function. Results: c-MET expression was required for Teliso-V cytotoxicity and intracellular payload delivery in all cell lines. Compared with WT-c-MET, various mutants showed oncogene-driven transformation evident by elevated levels of phosphorylated c-MET/AKT, cytokine-independent colony formation, oncogene-driven proliferation, and tyrosine kinase inhibitor (TKI) resistance. Interestingly, c-MET-TKD-expressing cells were the most sensitive to Teliso-V, which was associated with faster ADC internalization and EL trafficking. An AI-generated protein model and associated molecular dynamics simulation demonstrated that all oncogenic TKD variants tested stabilized the c-MET-plexin-semaphorin-integrin domain (R592-L614), known for its ability to position the extracellular ligand-binding (ELB) site for optimal activation. Conclusions: c-MET-TKD mutations induced TKI resistance and enhanced MMAE delivery in association with robust ADC internalization and EL trafficking. Previous studies have shown that the HER2 ADC fam-trastuzumab deruxtecan-nxki was highly effective in cell lines and patients with ERBB2 kinase domain-mutant lung cancer via a similar mechanism, raising the possibility that this may be a general phenomenon across receptor tyrosine kinase-targeting ADCs. c-MET-TKD mutations are predicted to stabilize the ELB conformation to activate enhanced oncogenic signaling that may contribute to rapid endocytosis-mediated drug delivery. Citation Format: Izhar S. Batth, Bijay S. Jaiswal, Dolonchampa Maji, Robert Sparks, William Glauser, Tamar Uziel, D. Ross Camidge, Athan Vasilopoulos, Peter Ansell, PK Epling-Burnette. c-MET mutations sensitize to antibody-drug conjugate telisotuzumab vedotin through efficient internalization and rapid intracellular drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 540.

Introduction: Venetoclax (Ven) is a selective, potent, oral BCL-2 inhibitor under investigation as a targeted therapy for t(11;14)-positive relapsed/refractory multiple myeloma (RRMM). The molecular mechanisms underlying the increased sensitivity of t(11;14) MM to Ven have not been fully elucidated; however, remnants of B-cell biology and reduced cellular bioenergetics have been associated with sensitivity to Ven in MM. Transcriptomic analysis of 302 RRMM patient tumor cells before treatment with Ven-based regimens, an ex vivo B-cell differentiation model, and metabolic profiling of 7 MM cell lines were assessed to understand the unique biology that distinguishes t(11;14) in MM. Methods: CD138-enriched bone marrow mononuclear cells were collected at baseline from multiple Ven clinical trials (NCT02755597, NCT01794520, NCT03314181, NCT02899052). Biomarker analyses, including BCL2 gene expression by quantitative PCR, t(11;14) status by interphase fluorescence in situ hybridization (FISH), and transcriptomic analysis by RNA sequencing (RNA-seq), were performed by a central laboratory. Isolated healthy memory B-cells (MBCs) were progressively differentiated ex vivo into pre-plasmablast (Pre-PB), plasmablast (PB), and plasma cells (PC). MM cell lines were differentiated by treatment with all-trans retinoic acid (ATRA) for 3-days. Global metabolomic profiling was performed by liquid chromatography-mass spectrometry (LC-MS) in MM cell lines with or without ATRA-mediated differentiation. Gene set variation analysis (GSVA) was used to establish B-cell and mitochondrial (Mito) gene signatures. Results: Consistent with previous findings, t(11;14)-positive RRMM expressed significantly higher CCND1 (mean log2 FPKM: 3.8 vs −0.4, P<.0001) and BCL2 levels (mean 2-ΔCt: 1.3 vs 0.4, P<.0001) compared with t(11;14)-negative RRMM. Using a network-based enrichment map to interrogate differentially expressed genes, transcriptomes of t(11;14)-positive samples were enriched in several pathways, including cell cycle, metabolism and biosynthesis, and immune pathways. Significantly higher B-cell signature scores, but lower Mito signature scores were detected in t(11;14)-positive versus t(11;14)-negative RRMM patients (P<.0001; Figure). The ex vivo B-cell differentiation model confirmed that B-cell and Mito scores are differentiation-stage specific and significantly changed upon differentiation to plasma cells (P<.0001). In RRMM patients, there was an inverse correlation between the B-cell and Mito signature scores (R=−0.6, P<.0001). In vitro models were then used to further investigate the relationship between B-cell differentiation and Ven sensitivity. ATRA differentiated t(11;14)-positive MM cell lines, as indicated by lower expression of CD20 and increased expression of the plasma cell markers CD138 and CD38, significantly reduced BCL2 expression and increased basal rates of respiration in association with increased mitochondrial mass, which correlated with reduced Ven sensitivity. Differentially expressed metabolites were overrepresented in the purine biosynthetic pathway in t(11;14)-positive MM cell lines, which was corroborated by differential gene expression within this pathway in the ex vivo B-cell differentiation model and in t(11;14)-positive RRMM patients. Using thresholds that balance sensitivity and specificity of the B-cell and Mito signature scores associated with t(11;14)-positive RRMM, progression-free survival (PFS) outcomes in the BELLINI study were not superior to those demonstrated by t(11;14) or high BCL2 expression status alone (Table). Conclusions: Using an integrated approach of transcriptomic and metabolomic profiling, t(11;14) MM cells were confirmed to retain features of B-cell biology, including lower Mito metabolism that results in increased sensitivity to BCL-2 inhibition by Ven. Although these biological features are closely linked to t(11;14), B-cell gene or Mito gene signatures did not serve as better biomarkers for predicting efficacy to Ven compared with t(11;14) alone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: Epcoritamab (Epco) is a CD3 bispecific antibody that induces selective, potent T-cell-mediated killing of CD20-positive malignant B-cells. In the phase 1/2 monotherapy study, subcutaneous administered Epco showed a favorable safety profile and promising efficacy, including complete responses in heavily pretreated patients with relapsed / refractory non-Hodgkin's lymphoma (Hutchings et al, Lancet 2021; Thieblemont et al, EHA 2022, LB2364; EPCORE NHL-1). Ongoing clinical studies combining Epco with standard-of-care therapies including combinations with Rituximab show promising preliminary anti-tumor activity in diffuse large B-cell lymphoma and follicular lymphoma (Falchi et al, ASCO 2022 abstracts 7523 and 7524; EPCORE NHL-2 arm 1 and 2; NCT04663347). Here we evaluated the competition for CD20 binding and functional interactions between these agents and modeled its impact on Epco, T-cell, and tumor cell trimer formation that drives Epco clinical activity. Aims: Investigate the preclinical binding and functional interaction between Epco and Rituximab to establish the potential for the combination of these agents in the treatment of B-cell NHL. Methods: Competition between Epco and Rituximab for binding to CD20 cells was assessed by Flow Cytometry after incubating the single labeled agent in the presence of increasing concentrations of the competitor and vice-versa (across 0.1 - 300 ug/ml evaluated for each agent), on a range of CD20 expressing lymphoma cell lines. Epco-induced-T cell activation and redirected T cell cytotoxicity was determined by co-culturing T cells with lymphoma cells with varying expression of CD20. Rituximab antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by incubating fresh peripheral blood mononuclear cells with lymphoma cells in the presence of a CD3 control Epco antibody (DuoBody-ctrlxCD20). Data from binding and cytotoxicity was used to quantify the degree of binding curve shifts at different Rituximab:Epco concentrations as well as the impact on predicted trimer formation and cytotoxicity, based on modeling. Results: In binding assays, the addition of Rituximab resulted in a dose-dependent reduction of Epco binding to CD20 expressing lymphoma cell lines, with a significant reduction of Epco binding observed at clinically relevant Epco concentrations in cells with high CD20 expression levels (Figure 1). Similar binding interference interactions were observed for binding of Epco to cell lines with low and intermediate CD20 expression levels. Binding of Rituximab to cell lines expressing high CD20 levels was not significantly reduced by clinically relevant concentrations of Epco. In Epco functional studies, the addition of increasing concentrations of Rituximab did not affect Epco cytotoxic activity at clinically relevant range of Rituximab doses tested (Figure 2). Similarly, no effect of Rituximab on Epco-mediated T cell activation was observed at clinically relevant concentrations of Rituximab and Epco. ADCC activity at clinically relevant concentrations of Rituximab (10-250 ug/ml) was not impacted by incubation with the CD3 control Epco antibody (DuoBody-ctrlxCD20), even at concentrations exceeding the Epco clinical concentrations. Modeling and simulation of in vitro data showed that at clinically relevant concentration of Epco, the presence of clinically relevant concentration of Rituximab does not meaningfully impact Epco trimer formation, and the predicted trimer formation remains above EC90 relative to that needed for 50% cell kill. Conclusions: Our studies showed that while Rituximab exerts significant interference on Epco binding to CD20-expressing cells, tumor-cell cytotoxicity by Epco is minimally affected by clinically relevant concentrations of Rituximab. This conclusion is supported by preclinical modeling and simulations showing that Epco activity driven by trimer formation is not meaningfully impacted by clinical concentrations of Rituximab. Furthermore, we found a limited effect of Epco on Rituximab binding to CD20 and no impact on ADCC activity by Rituximab, thereby supporting potential synergy in the combination between these agents. Results from this study confirm the ability to combine Epco with CD20 antibodies, specifically with Rituximab, and the potential for synergistic activity of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CMML is a lethal myeloid neoplasm with no therapies that improve its dismal prognosis. Inhibition of BET family members has been proposed as a therapeutic strategy based on preclinical data identifying BRD4 as a therapeutic target in acute myeloid leukemia. However, despite potent on-target transcriptional remodeling, early phase clinical trials have demonstrated only modest activity secondary to a variety of resistance mechanisms. In ovarian cancer BET inhibitor (BETi) treated cells, compensatory upregulation and addiction to pro-survival kinase networks have been observed. Given that over 50% of CMML cases have mutations upregulating kinase signaling, we hypothesized that BETi resistance is mediated by these networks in CMML and can be targeted therapeutically. We tested this hypothesis by performing a limited screen of kinase inhibitors alone and in combination with the IC20 of the BETi INCB54329 in 8 human leukemia cell lines. This screen revealed that the IC50 of the PIM inhibitor (PIMi) INCB53914 decreased after co-treatment with BETi in a majority of the leukemia cell lines tested. Synergy was validated chemically in U937, TF1 and SKM1 leukemia cells using other selective inhibitors of BET and PIM. We next assessed the activity of the BET-PIM combination in 14-day colony formation assays with 10 unique CMML bone marrow mononuclear cell (BM-MNCs) patient samples(Fig. 1A). These studies revealed that combination therapy significantly suppressed clonogenicity versus BMNCs treated with vehicle or single drug alone. Finally, this synergy was validated in vivo in 36 patient derived xenografts (PDX) from 3 CMML patients, as manifest by reduced leukemic burden/engraftment in CMML PDX treated with combination therapy(Fig. 1B). To explore the mechanism by which BETi and PIMi therapeutically synergize we treated U937 and SKM1 leukemia cells with INCB54329 and measured mRNA and protein levels for all PIM isoforms. Surprisingly, we identified that PIM1 was increased following treatment with INCB54329, other BETi, or a JQ1-derived PROTAC (Fig. 1C). PIM1 upregulation was also manifest in INCB54329 persistor U937 leukemia cells generated by daily BETi treatment for 6 weeks. Testing across a broader panel of leukemia cell lines revealed an inverse correlation between PIM1 induction and decrease in the IC50 of PIMi following BETi treatment, suggesting PIM1 upregulation confers sensitivity to combination therapy. Consistent with this, isogenic SKM1 leukemia cells engineered to overexpress PIM1 were resistant to INCB54329 and were more sensitive to INCB53914 versus controls cells. Recent studies have demonstrated that inhibitory miRNAs, especially those located near super-enhancers, are suppressed by BET inhibition. Given that several miRNAs are known to control PIM1 expression, we hypothesized that paradoxical PIM1 upregulation following BETi treatment was due to down-regulation of select miRNAs. To test this, we treated our leukemia cell models with broad inhibitors of miRNA activity (i.e., AGO and Dicer inhibitors) and observed a dose dependent increase in PIM1 levels similar to that seen with BET inhibition(Fig. 1Di). Further, integrating public H3K27 CHIP-seq and miRNA super enhancer datasets and using computational prediction algorithms, we identified 6 candidate miRNAs that could regulate PIM1 and were predicted to be controlled by BET inhibitors. Of these, only miR-33a levels were reduced in a dose dependent manner in SKM1 cells by BETi treatment(Fig. 1Dii). This was confirmed by genetically silencing all BET proteins, which suppressed miR-33a levels in SKM1 leukemia cells. Finally, miR-33a mimics (but not control miRNAs) abolished BETi-induced upregulation of PIM1(Fig. 1Diii). Collectively, these studies established BET and PIM inhibition as a novel and potent combination therapy for CMML that is mediated by miR-33a-dependent upregulation of PIM1(Fig. 1E). Disclosures Liu: Incyte Corporation: Employment. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding. Komrokji:Novartis: Speakers Bureau; JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy. Epling-Burnette:Incyte Corporation: Research Funding. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Haura:Incyte Corporation: Research Funding. Reuther:Incyte Corporation: Research Funding. Koblish:Incyte Corporation: Employment.

Approximately three million non-melanoma skin cancers (NMSCs) are diagnosed worldwide each year, although this number is likely an underestimate given that these cancers are not always recorded in cancer registries. Studies have suggested that skin (cutaneous) infections with human papillomaviruses (HPV) and polyomaviruses (HPyV) may play a role in the development of some NMSC types. Suppression of the immune system is also a risk factor for NMSC. This study, from the U.S.A, aimed to understand the relationship between T-regulatory (Treg) cells, cells which suppress immune response, and cutaneous viral infections. Blood, skin swabs and eyebrow hairs were collected from 352 patients who underwent skin cancer screening and did not have cancer detected. The researchers examined whether the skin swabs (SSW) and eyebrow hairs (EBH) contained genetic material (DNA) corresponding to 98 cutaneous HPV types (including beta HPV and gamma HPV) and 5 HPyV types. The blood samples were analyzed to determine proportions of different types of Treg cell populations in circulation (in the blood). The researchers found no association between total percent of circulating Treg cells and beta HPV or HPyV infection. However, two types of Treg cells were found at lower levels in those that had gamma HPV infection in their EBH and/or SSW. Those two types were CLA + Treg cells (known to travel to the skin) and effector CD27 - CD45RA - FOXP3 + CD4 + Treg cells (known to become active when exposed to a foreign viral infection). The study results suggest that gamma HPV infection may stimulate Treg cells to move from circulation into the skin tissues.

每年全世界约诊断出300万例非黑色素瘤皮肤癌(NMSC),尽管这个数字很可能被低估了,考虑到这类癌症不总是被记录在癌症注册数据库中。研究表明,人乳头瘤病毒 (HPV)和多瘤病毒(HPyV)皮肤感染可能对某些类型NMSC的出现起作用。免疫系统的抑制也是NMSC的一个风险因素。这项美国的研究旨在了解调节性T‐细胞(Treg)(抑制免疫反应的细胞)和病毒性皮肤感染之间的关系。从352名进行皮肤癌筛查且未检测到癌症的患者中收集血液、皮肤拭子和眉毛。研究者分析了皮肤拭子 (SSW) 和眉毛 (EBH) 是否含有 98 种皮肤 HPV(包括 β HPV 和 γ HPV)及 5 种 HPyV 相应的基因物质 (DNA)。还分析了血液样本,以确定在血液循环中不同类型T细胞群体的比例。研究发现,在血液循环 T 细胞的总比例和 β HPV 或 HPyV 感染之间无相关性。但发现在 EBH 和/或 SSW 出现 γ HPV 感染的患者中两种 T 细胞的水平更低。这两种 T细胞是 CLA+ T 细胞(已知可转运至皮肤)和CD27‐CD45RA‐FOXP3+CD4+效应T细胞(已知在暴露于外来病毒感染时激活)。研究结果表明,γHPV感染可能会刺激 T 细胞从血液循环转运至皮肤组织。

Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte–macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.

Background: The EZH2 gene (Enhancer of Zester homolog 2), located on chromosome (chr) 7q, is mutated in ~10% of patients with myelodysplastic syndromes (MDS) or MDS/myeloproliferative neoplasm (MDS/MPN). EZH2 gene mutations are associated with negative clinical outcomes; however, the role of EZH2 protein in MDS or MDS/MPN is largely unknown. In contrast to lymphomas or solid tumors in which expression is upregulated, decreased EZH2 mRNA expression was recently reported in 47% of MDS patients compared to normal controls and was more prevalent in patients with chr7 abnormalities. Furthermore, patients with decreased EZH2 expression lacking chr7 abnormalities had better overall survival (OS) (Cabrero M et al 2016). This study aims to explore EZH2 protein expression by immunohistochemistry (IHC) in MDS and MDS/MPN and the relationship to gene mutation and clinical outcome.