SUSD2 suppresses CD8+ T cell antitumor immunity by targeting IL-2 receptor signaling.

Dysfunctional CD8+ T cells, which have defective production of antitumor effectors, represent a major mediator of immunosuppression in the tumor microenvironment. Here, we show that SUSD2 is a negative regulator of CD8+ T cell antitumor function. Susd2-/- effector CD8+ T cells showed enhanced production of antitumor molecules, which consequently blunted tumor growth in multiple syngeneic mouse tumor models. Through a quantitative mass spectrometry assay, we found that SUSD2 interacted with interleukin (IL)-2 receptor α through sushi domain-dependent protein interactions and that this interaction suppressed the binding of IL-2, an essential cytokine for the effector functions of CD8+ T cells, to IL-2 receptor α. SUSD2 was not expressed on regulatory CD4+ T cells and did not affect the inhibitory function of these cells. Adoptive transfer of Susd2-/- chimeric antigen receptor T cells induced a robust antitumor response in mice, highlighting the potential of SUSD2 as an immunotherapy target for cancer.

DLL3-guided therapies in small-cell lung cancer: from antibody-drug conjugate to precision immunotherapy and radioimmunotherapy.

DLL3 acts as an inhibitory ligand that downregulates Notch signaling and is upregulated by ASCL1, a transcription factor prevalent in the small-cell lung cancer (SCLC) subtype SCLC-A. Currently, the therapeutic strategies targeting DLL3 are varied, including antibody-drug conjugates (ADCs), bispecific T-cell engagers (BiTEs), and chimeric antigen receptor (CAR) T-cell therapies. Although rovalpituzumab tesirine (Rova-T) showed promise in a phase II study, it failed to produce favorable results in subsequent phase III trials, leading to the cessation of its development. Conversely, DLL3-targeted BiTEs have garnered significant clinical interest. Tarlatamab, for instance, demonstrated enhanced response rates and progression-free survival compared to the standard of care in a phase II trial; its biologics license application (BLA) is currently under US Food and Drug Administration (FDA) review. Numerous ongoing phase III studies aim to further evaluate tarlatamab's clinical efficacy, alongside the development of novel DLL3-targeted T-cell engagers, both bispecific and trispecific. CAR-T cell therapies targeting DLL3 have recently emerged and are undergoing various preclinical and early-phase clinical studies. Additionally, preclinical studies have shown promising efficacy for DLL3-targeted radiotherapy, which employs ß-particle-emitting therapeutic radioisotopes conjugated to DLL3-targeting antibodies. DLL3-targeted therapies hold substantial potential for SCLC management. Future clinical trials will be crucial for comparing treatment outcomes among various approaches and exploring combination therapies to improve patient survival outcomes.

Integrative comparison of the genomic and transcriptomic landscape between prostate cancer patients of predominantly African or European genetic ancestry.

Men of predominantly African Ancestry (AA) have higher prostate cancer (CaP) incidence and worse survival than men of predominantly European Ancestry (EA). While socioeconomic factors drive this disparity, genomic factors may also contribute to differences in the incidence and mortality rates. To compare the prevalence of prostate tumor genomic alterations and transcriptomic profiles by patient genetic ancestry, we evaluated genomic profiles from The Cancer Genome Atlas (TCGA) CaP cohort (n = 498). Patient global and local genetic ancestry were estimated by computational algorithms using genotyping data; 414 (83.1%) were EA, 61 (12.2%) were AA, 11 (2.2%) were East Asian Ancestry (EAA), 10 (2.0%) were Native American (NA), and 2 (0.4%) were other ancestry. Genetic ancestry was highly concordant with self-identified race/ethnicity. Subsequent analyses were limited to 61 AA and 414 EA cases. Significant differences were observed by ancestry in the frequency of SPOP mutations (20.3% AA vs. 10.0% EA; p = 5.6×10-03), TMPRSS2-ERG fusions (29.3% AA vs. 39.6% EA; p = 4.4×10-02), and PTEN deletions/losses (11.5% AA vs. 30.2% EA; p = 3.5×10-03). Differentially expressed genes (DEGs) between AAs and EAs showed significant enrichment for prostate eQTL target genes (p = 8.09×10-48). Enrichment of highly expressed DEGs for immune-related pathways was observed in AAs, and for PTEN/PI3K signaling in EAs. Nearly one-third of DEGs (31.3%) were long non-coding RNAs (DE-lncRNAs). The proportion of DE-lncRNAs with higher expression in AAs greatly exceeded that with lower expression in AAs (p = 1.2×10-125). Both ChIP-seq and RNA-seq data suggested a stronger regulatory role for AR signaling pathways in DE-lncRNAs vs. non-DE-lncRNAs. CaP-related oncogenic lncRNAs, such as PVT1, PCAT1 and PCAT10/CTBP1-AS, were found to be more highly expressed in AAs. We report substantial heterogeneity in the prostate tumor genome and transcriptome between EA and AA. These differences may be biological contributors to racial disparities in CaP incidence and outcomes.

Factors Affecting the Cancer Immunotherapeutic Efficacy of T Cell Bispecific Antibodies and Strategies for Improvement.

T-cell bispecific antibodies (T-BsAbs) are a new class of cancer immunotherapy drugs that can simultaneously bind to tumor-associated antigens on target cells and to the CD3 subunit of the T-cell receptor (TCR) on T cells. In the last decade, numerous T-BsAbs have been developed for the treatment of both hematological malignancies and solid tumors. Among them, blinatumomab has been successfully used to treat CD19 positive malignancies and has been approved by the FDA as standard care for acute lymphoblastic leukemia (ALL). However, in many clinical scenarios, the efficacy of T-BsAbs remains unsatisfactory. To further improve T-BsAb therapy, it will be crucial to better understand the factors affecting treatment efficacy and the nature of the T-BsAb-induced immune response. Herein, we first review the studies on the potential mechanisms by which T-BsAbs activate T-cells and how they elicit efficient target killing despite suboptimal costimulatory support. We focus on analyzing reports from clinical trials and preclinical studies, and summarize the factors that have been identified to impact the efficacy of T-BsAbs. Lastly, we review current and propose new approaches to improve the clinical efficacy of T-BsAbs.

Prognostic significance of translocations in the presence of mutated IGHV and of cytogenetic complexity at diagnosis of chronic lymphocytic leukemia.

Mutations of the IGH variable region in patients with chronic lymphocytic leukemia (CLL) are associated with a favorable prognosis. Cytogenetic complexity (>3 unrelated aberrations) and translocations have been associated with an unfavorable prognosis. While mutational status of IGHV is stable, cytogenetic aberrations frequently evolve. However, the relationships of these features as prognosticators at diagnosis are unknown. We examined the CpG-stimulated metaphase cytogenetic features detected within one year of diagnosis of CLL and correlated these features with outcome and other clinical features including IGHV. Of 329 untreated patients, 53 (16.1%) had a complex karyotype (16.1%), and 85 (25.8%) had a translocation. Median time to first treatment (TFT) was 47 months. In univariable analyses, significant risk factors for shorter TFT (p3.5, log-transformed WBC, unmutated IGHV, complex karyotype, translocation, and FISH for trisomy 8, del(11q) and del(17p). In multivariable analysis, there was significant effect modification of IGHV status on the relationship between translocation and TFT (p=0.002). In IGHV mutated patients, those with a translocation had over 3.5 times higher risk of starting treatment than those without a translocation (p.

T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells.

Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T cell receptor (TCR)γδ(+) T cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T cells by Treg cells maintains enteric immune tolerance.

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

Nuclear factor-kappaB modulates regulatory T cell development by directly regulating expression of Foxp3 transcription factor.

Naturally derived regulatory T (Treg) cells are characterized by stable expression of the transcription factor Foxp3 and characteristic epigenetic imprinting at the Foxp3 gene locus. Here, we found that enhancing nuclear factor (NF)-kappaB activity via a constitutive active inhibitor of kappaB kinase beta (IKKbeta) transgene in T cells led to increased number of Foxp3(+) cells in the thymus and can rescue Foxp3 expression in thymocytes deficient in other pleiotropic signaling molecules. Enhancing the signal strength of the NF-kappaB pathway also induced Foxp3 expression in otherwise conventionally selected T cells. NF-kappaB directly promoted the transcription of Foxp3, and upon T cell receptor (TCR) stimulation, c-Rel, a NF-kappaB family member, bound to Foxp3 enhancer region, which is specifically demethylated in natural Treg cells. Hence, NF-kappaB signaling pathway is a key regulator of Foxp3 expression during natural Treg cell development.

Differential role of the transcription factor NF-kappaB in selection and survival of CD4+ and CD8+ thymocytes.

Inhibition of the transcription factor nuclear factor (NF)-kappaB activity leads to a reduction in numbers of CD8(+) single-positive (SP) thymocytes, suggesting a selective role for NF-kappaB in these cells. To further explore the role of NF-kappaB in SP thymocytes, we utilized transgenic models that allowed either inhibition or activation of NF-kappaB. We showed that activation of NF-kappaB played an important role in the selection of major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. Surprisingly, NF-kappaB was not activated in positively selected CD4(+) thymocytes, and inhibition of NF-kappaB did not perturb positive or negative selection of CD4(+) cells. However, enforced activation of NF-kappaB via a constitutively active inhibitor of kappaB (IkappaB) kinase transgene led to a nearly complete deletion of CD4 cells by pushing positively selecting CD4(+) cells into negative selection. These studies therefore revealed a surprising difference of NF-kappaB activation in CD4(+) and CD8(+) thymocytes and suggested that NF-kappaB contributes to the establishment of thresholds of signaling that determine positive or negative selection of thymocytes.

IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response.

The activation of pro-inflammatory gene programs by nuclear factor-kappaB (NF-kappaB) is primarily regulated through cytoplasmic sequestration of NF-kappaB by the inhibitor of kappaB (IkappaB) family of proteins. IkappaBbeta, a major isoform of IkappaB, can sequester NF-kappaB in the cytoplasm, although its biological role remains unclear. Although cells lacking IkappaBbeta have been reported, in vivo studies have been limited and suggested redundancy between IkappaBalpha and IkappaBbeta. Like IkappaBalpha, IkappaBbeta is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus. The crystal structure of IkappaBbeta bound to p65 suggested this complex might bind DNA. In vitro, hypophosphorylated IkappaBbeta can bind DNA with p65 and c-Rel, and the DNA-bound NF-kappaB:IkappaBbeta complexes are resistant to IkappaBalpha, suggesting hypophosphorylated, nuclear IkappaBbeta may prolong the expression of certain genes. Here we report that in vivo IkappaBbeta serves both to inhibit and facilitate the inflammatory response. IkappaBbeta degradation releases NF-kappaB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-alpha (TNF-alpha). Surprisingly, absence of IkappaBbeta results in a dramatic reduction of TNF-alpha in response to LPS even though activation of NF-kappaB is normal. The inhibition of TNF-alpha messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IkappaBbeta bound to p65:c-Rel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaBbeta acts through p65:c-Rel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaBbeta(-/-) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IkappaBbeta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha production during the inflammatory response.

A CD3 humanized mouse model unmasked unique features of T-cell responses to bispecific antibody treatment.

ABSTRACT: T-cell bispecific antibodies (T-BsAbs) such as blinatumomab hold great promise for cancer immunotherapy. A better understanding of the in vivo immune response induced by T-BsAbs is crucial to improving their efficacy and safety profile. However, such efforts are hindered by the limitations of current preclinical models. To address this, we developed a syngeneic murine model with humanized CD3 and target antigen (CD20). This model enables the development of disseminated leukemia with a high tumor burden, which mirrors clinical findings in human patients with relapsed/refractory acute lymphoblastic leukemia. Treatment of this model with T-BsAbs results in cytokine release syndrome, with cytokine profiles and levels reflecting observations made in human patients. This model also faithfully recapitulates the dynamics of T-cell activation seen in human patients, including the temporary disappearance of T cells from the bloodstream. During this phase, T cells are sequestered in secondary lymphoid organs and undergo activation. Clinical correlative studies that rely primarily on peripheral blood samples are likely to overlook this critical activation stage, leading to a substantial underestimation of the extent of T-cell activation. Furthermore, we demonstrate that surface expression of the T-BsAb target antigen by leukemia cells triggers a swift immune response, promoting their own rejection. Humanizing the target antigen in the recipient mice is crucial to facilitate tolerance induction and successful establishment of high tumor burden. Our findings underscore the importance of meticulously optimized syngeneic murine models for investigating T-BsAb-induced immune responses and for translational research aimed at improving efficacy and safety.

Siglec-6 as a therapeutic target for cell migration and adhesion in chronic lymphocytic leukemia.

Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.

Repression of PRMT activities sensitize homologous recombination-proficient ovarian and breast cancer cells to PARP inhibitor treatment.

An "induced PARP inhibitor (PARPi) sensitivity by epigenetic modulation" strategy is being evaluated in the clinic to sensitize homologous recombination (HR)-proficient tumors to PARPi treatments. To expand its clinical applications and identify more efficient combinations, we performed a drug screen by combining PARPi with 74 well-characterized epigenetic modulators that target five major classes of epigenetic enzymes. Both type I PRMT inhibitor and PRMT5 inhibitor exhibit high combination and clinical priority scores in our screen. PRMT inhibition significantly enhances PARPi treatment-induced DNA damage in HR-proficient ovarian and breast cancer cells. Mechanistically, PRMTs maintain the expression of genes associated with DNA damage repair and BRCAness and regulate intrinsic innate immune pathways in cancer cells. Analyzing large-scale genomic and functional profiles from TCGA and DepMap further confirms that PRMT1, PRMT4, and PRMT5 are potential therapeutic targets in oncology. Finally, PRMT1 and PRMT5 inhibition act synergistically to enhance PARPi sensitivity. Our studies provide a strong rationale for the clinical application of a combination of PRMT and PARP inhibitors in patients with HR-proficient ovarian or breast cancer.

Androgen ablation mitigates tolerance to a prostate/prostate cancer-restricted antigen.

To understand the T cell response to prostate cancer, we created transgenic mice that express a model antigen in a prostate-restricted pattern and crossed these animals to TRAMP mice that develop spontaneous prostate cancer. Adoptive transfer of prostate-specific CD4 T cells shows that, in the absence of prostate cancer, the prostate gland is mostly ignored. Tumorigenesis allows T cell recognition of the prostate gland--but this recognition is tolerogenic, resulting in abortive proliferation and ultimately in hyporesponsiveness at the systemic level. Androgen ablation (the most common treatment for metastatic prostate cancer) was able to mitigate this tolerance--allowing prostate-specific T cells to expand and develop effector function after vaccination. These results suggest that immunotherapy for prostate cancer may be most efficacious when administered after androgen ablation.

Systematic illumination of druggable genes in cancer genomes.

By combining 6 druggable genome resources, we identify 6,083 genes as potential druggable genes (PDGs). We characterize their expression, recurrent genomic alterations, cancer dependencies, and therapeutic potentials by integrating genome, functionome, and druggome profiles across cancers. 81.5% of PDGs are reliably expressed in major adult cancers, 46.9% show selective expression patterns, and 39.1% exhibit at least one recurrent genomic alteration. We annotate a total of 784 PDGs as dependent genes for cancer cell growth. We further quantify 16 cancer-related features and estimate a PDG cancer drug target score (PCDT score). PDGs with higher PCDT scores are significantly enriched for genes encoding kinases and histone modification enzymes. Importantly, we find that a considerable portion of high PCDT score PDGs are understudied genes, providing unexplored opportunities for drug development in oncology. By integrating the druggable genome and the cancer genome, our study thus generates a comprehensive blueprint of potential druggable genes across cancers.

Systematic Pan-Cancer Characterization of Nuclear Receptors Identifies Potential Cancer Biomarkers and Therapeutic Targets.

The nuclear receptor (NR) superfamily is one of the major druggable gene families, representing targets of approximately 13.5% of approved drugs. Certain NRs, such as estrogen receptor and androgen receptor, have been well demonstrated to be functionally involved in cancer and serve as informative biomarkers and therapeutic targets in oncology. However, the spectrum of NR dysregulation across cancers remains to be comprehensively characterized. Through computational integration of genetic, genomic, and pharmacologic profiles, we characterized the expression, recurrent genomic alterations, and cancer dependency of NRs at a large scale across primary tumor specimens and cancer cell lines. Expression levels of NRs were highly cancer-type specific and globally downregulated in tumors compared with corresponding normal tissue. Although the majority of NRs showed copy-number losses in cancer, both recurrent focal gains and losses were identified in select NRs. Recurrent mutations and transcript fusions of NRs were observed in a small portion of cancers, serving as actionable genomic alterations. Analysis of large-scale CRISPR and RNAi screening datasets identified 10 NRs as strongly selective essential genes for cancer cell growth. In a subpopulation of tumor cells, growth dependencies correlated significantly with expression or genomic alterations. Overall, our comprehensive characterization of NRs across cancers may facilitate the identification and prioritization of potential biomarkers and therapeutic targets, as well as the selection of patients for precision cancer treatment. SIGNIFICANCE: Computational analysis of nuclear receptors across multiple cancer types provides a series of biomarkers and therapeutic targets within this protein family.

Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.