Justin P. Annes

Succinate dehydrogenase B ( SDHB )-deficient cancers, including pheochromocytoma and paraganglioma (PPGL), renal cell carcinoma (RCC), and gastrointestinal stromal tumor (GIST), exhibit aggressive clinical behavior with limited treatment options. To identify novel therapeutic vulnerabilities, we performed high-throughput drug screening using co-cultured human SDHB -deficient and SDHB -reconstituted UOK269 human renal cell carcinoma cells with dual fluorescence labeling. Several compounds displaying preferential cytotoxicity toward SDHB -deficient cells were known substrates of the ATP-binding cassette B1 (ABCB1) transporter, also known as Multidrug Resistance Protein 1 (MDR1). Accordingly, SDHB -deficient cells demonstrated markedly reduced ABCB1 expression, a phenotype confirmed across human SDHB -deficient PPGL, RCC, and GIST samples, but absent in IDH - or FH -deficient tumors. Mechanistic investigation revealed that KDM4A histone demethylase is downregulated in SDHB -deficient tumors, and KDM4A loss (or inhibition) is sufficient to reduce ABCB1 expression, globally increase H3K9me3 levels, and sensitize cells to cytotoxic ABCB1 substrates. TCGA analysis confirmed correlated KDM4A-ABCB1 expression in SDHB -deficient cancers, with ABCB1 levels predicting drug sensitivity across cancer cell lines. Clinical analysis demonstrated that SDHB -mutant PPGL patients treated with vincristine-containing chemotherapy exhibited significantly longer progression-free survival compared to non- SDHB patients. These findings establish a novel metabolic-epigenetic pathway linking SDHB deficiency to ABCB1 silencing and provide rational basis for repurposing vincristine, and related ABCB1 substrate vinca alkaloids, across SDHB -deficient cancer types; offering an alternative precision treatment approach for these challenging malignancies.

Type 1 diabetes (T1D) is caused by T cell-mediated autoimmune destruction of insulin-producing islet beta-cells. Treatment with T-cell depleting therapies delays the progression of stage 2 and 3 T1D, but these agents exert broad immunosuppressive effects on T cell populations, including T regulatory cells (Tregs), which are key in promoting immune tolerance. We evaluated non-obese diabetic (NOD) mice and recently diagnosed T1D patients and identified CD38 as a marker for pathogenic T cell populations. Using adoptive T-cell transfer in Recombination Activating Gene 1 knockout NOD mice and in a humanized mouse model of autoimmune diabetes, we demonstrated that CD38-expressing autoreactive T cells drive diabetes pathogenesis. Furthermore, we found that selective depletion of CD38+ cells, using an anti-CD38 monoclonal antibody (mAb), prevents insulitis and diabetes onset without depleting CD4+CD25+ Tregs. Administration of anti-CD38 mAb did not adversely affect islet function and may selectively eliminate immunogenic senescent islet beta-cells. These results support the strategy of selectively depleting diabetogenic T cells using an anti-CD38 mAb to treat T1D and restore immune tolerance. Therefore, transient depletion of autoreactive T cells using anti-CD38 mAb may provide a novel strategy to prevent or abrogate autoimmunity in T1D.

The hereditary pheochromocytoma and paraganglioma (hPPGL) syndrome, caused by germline mutations in succinate dehydrogenase (SDHx) genes, predisposes individuals to pheochromocytomas (Pheo), paragangliomas (PGLs), renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GISTs). Notably, tumors with succinate dehydrogenase subunit B (SDHB) deficiency demonstrate an increased metastatic risk and current systemic treatments remain only palliative. Hence, discovering novel therapeutic avenues to improve SDHB cancer prognosis is an urgent need. Here, we leveraged human SDHB-deficient UOK269 RCC cells (SDHB-KO) and isogenic SDHB-reconstituted control cells (SDHB-WT) to discover SDH-dependent mitochondria-directed cytotoxic agents. Given the reduced ATP-generating capacity of SDHB-KO cells, we hypothesized that they would be uniquely sensitive to futile cycle induction with mitochondrial ionophores. Indeed, ionophores exhibited preferential cytotoxicity toward SDHB-KO cells. However, the mitochondria-directed chemotherapeutic compound Ym155 demonstrated more potent and dramatic preferential cytotoxicity toward SDHB-KO cells. Importantly, SDH-dependent cytotoxicity of Ym155 was validated in multiple cell models, including primary human pheochromocytoma cells, a mouse pheochromocytoma (MPC) cell line and primary SDHB-deficient mouse kidney cells. Notably, genetic evidence of Ym155 synthetic lethality with SDHB deficiency was buttressed in additional cell models using two chemical inhibitors of SDH enzyme activity. Mechanistically, SDH deficiency sensitized cells to Ym155-induced DNA damage. Strikingly, SDH-dependent Ym155 sensitivity was recapitulated by inhibition of the histone demethylase KDM4, a downstream consequence of SDH deficiency. In summary, accumulation of succinate in SDH-deficient tumors inhibited KDM4 activity, impaired DNA repair and yielded enhanced susceptibility to Ym155-induced reactive oxygen species (ROS) generation. The identified intrinsic susceptibilities of SDHB-deficient cancers have the potential to be therapeutically leveraged.

Pseudopheochromocytoma is a disorder characterized by paroxysmal hypertension and variably elevated catecholamine metabolite levels. Pseudopheochromocytoma clinically mimics pheochromocytoma but differs in etiology. While pheochromocytoma is a catecholamine-secreting neuroendocrine tumor, pseudopheochromocytoma is a syndrome linked to a history of emotional stressors and is believed to stem from autonomic nervous system dysregulation. We present the case of a 70-year-old female patient experiencing episodic hypertensive crises for 3 decades. The patient was referred to endocrine oncology for evaluation of a possible pheochromocytoma due to her long-standing history of symptomatic hypertension and elevated catecholamine metabolites. Anatomic and functional imaging, including computed tomography scans of the abdomen and pelvis and a 64Copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-octreotate positron emission tomography computed tomography excluded a diagnosis of pheochromocytoma or paraganglioma. Her history of significant emotional stressors raised the possibility of pseudopheochromocytoma. Following initiation of escitalopram and psychotherapy, the patient experienced a remarkable improvement in the frequency and severity of hypertensive episodes. This case illustrates the diagnostic challenges of pseudopheochromocytoma and the importance of early intervention in preventing complications.

Hereditary pheochromocytoma and paraganglioma (hPPGL) is caused by pathogenic mutations in succinate dehydrogenase (SDH) genes, commonly SDHB . However, over 80% of SDHB missense variants are classified as variants of uncertain significance (VUS), limiting clinical interpretation and diagnostic utility of germline testing. To provide functional evidence of SDHB allele pathogenicity or benignity, we developed a cellular complementation assay that quantifies intracellular succinate/fumarate ratios as a readout of SDH enzymatic activity. This assay reliably distinguished pathogenic from benign alleles with high fidelity, outperforming and complementing computational predictions. Functional assessment of patient-derived VUS alleles supported reclassification of 87% of tested variants and revealed that mutations in the iron-sulfur cluster domain were amorphic, while those at or beyond the C-terminal residue Tyr273 retained function. Variants associated with Leigh syndrome retained activity, consistent with their biallelic inheritance and distinct pathogenic mechanisms from SDHB -related tumorigenesis. Notably, hypomorphic pathogenic SDHB variants correlated with increased head and neck paraganglioma occurrence, revealing a genotype-phenotype relationship. Functional characterization of SDHB missense variants supports clinical classification, informs hPPGL risk stratification, and has immediate diagnostic impact.

The hereditary pheochromocytoma and paraganglioma (hPPGL) syndrome is caused by inherited mutations in Succinate Dehydrogenase genes (SDHx). Affected individuals are predisposed to developing pheochromocytomas (Pheo), paragangliomas (PGL), renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GIST). Notably, tumors with succinate dehydrogenase subunit B ( SDHB ) deficiency demonstrate increased metastatic risk, for which treatments remain palliative. Hence, discovering novel therapeutic avenues to improve the prognosis for SDHB -cancer patients is an urgent need. Here we employed human SDHB -deficient UOK269 RCC cells ( SDHB -KO) and isogenic SDHB -reconstituted control cells ( SDHB -WT) to discover SDH-dependent mitochondria-directed cytotoxic agents. Given the reduced ATP-generating capacity of SDHB -KO cells, we hypothesized they would be uniquely sensitive to futile cycle induction with mitochondrial ionophores (2,4-Dinitrophenol (2-DNP), BAM15, Niclosamide, Nitazoxanide). Indeed, these compounds exhibited preferential cytotoxicity toward SDHB -KO cells. However, the chemotherapeutic compound Ym155 demonstrated the most potent and dramatic (five-fold) preferential cytotoxicity towards SDHB -KO cells. Importantly, the SDH-dependent cytotoxicity of Ym155 was validated in both primary human pheochromocytoma cells and mouse pheochromocytoma (MPC) cells. Furthermore, because few SDH-deficient cell lines are available, we buttressed our findings in additional relevant cell lines by modeling SDH-deficiency using chemical SDH enzyme inhibition with 3-nitropropionic acid (3-NPA). We observed a persistent cooperativity between SDH-deficiency and Ym155 cytotoxicity across multiple cell lineages and disease models. Mechanistically, Ym155-induced cytotoxicity was independent of its primary target, Survivin. Instead, SDH-deficiency sensitized cells to Ym155-induced DNA damage. Strikingly, the phenotype of SDH-deficient Ym155 sensitivity was recapitulated by inhibition of the histone demethylase KDM4, a downstream consequence of SDH deficiency. Thus, the accumulation of succinate in SDH-deficient tumors inhibits KDM4 activity, impairs DNA repair and yields enhanced susceptibility to Ym155-induced reactive oxygen species (ROS) generation. The identified intrinsic susceptibilities of SDHB -deficient cancers has the potential to be therapeutically leveraged.

Prolactin-secreting pituitary adenomas are typically treated with dopamine agonists to inhibit prolactin secretion and reduce tumor size. Dopamine-secreting paragangliomas are rare neuroendocrine tumors of sympathetic and parasympathetic paraganglia and often do not provoke symptoms of catecholamine excess. Although overlapping genetic drivers have been described for paragangliomas and pituitary adenomas, biochemical crosstalk between coexisting tumors is underexplored. We describe the case of a 52-year-old male individual who presented with cerebrospinal fluid (CSF) rhinorrhea and was found to have an invasive, 4.2-cm pituitary mass with modestly elevated prolactin (130.9 ng/mL [130.9 µg/L], reference range [RR] 2-18 ng/mL [2-18 µg/L]). Additional imaging discovered a mediastinal mass suspicious for a thoracic paraganglioma. Biochemical screening demonstrated marked elevation of plasma and urinary dopamine. Following paraganglioma resection, dopamine levels normalized, but prolactin rose significantly (877.8 ng/mL [877.8 µg/L]), suggesting an endogenous dopamine agonist–like effect from the paraganglioma to suppress pituitary prolactin hypersecretion. Pituitary pathology was notable for a PIT1 (pituitary transcription factor-1)-lineage pituitary adenoma with absent immunohistochemical staining for prolactin. Genetic testing found a previously unreported germline SDHC variant of uncertain significance. In this case, we report a novel biologic signaling mechanism between 2 rare primary endocrine tumors and highlight challenges in their diagnosis and management.

Cadaveric islet and stem cell–derived transplantation hold promise as treatments for type 1 diabetes. To tackle the issue of immunocompatibility, numerous cellular macroencapsulation techniques that use diffusion to transport insulin across an immunoisolating barrier have been developed. However, despite several devices progressing to human clinical trials, none have successfully attained physiological glucose control or insulin independence. Based on empirical evidence, macroencapsulation methods with multilayered, high islet surface density are incompatible with on-demand insulin delivery and physiological glucose regulation when solely reliant on diffusion. An additional driving force is essential to overcome the distance limit of diffusion. In this study, we present both theoretical evidence and experimental validation that applying pressure, at levels comparable to physiological diastolic blood pressure, significantly enhances insulin flux across immunoisolation membranes, increasing it by nearly three orders of magnitude. This significant enhancement in transport rate allows for precise, subminute regulation of both bolus and basal insulin delivery. By incorporating this technique with a pump-based extravascular system, we demonstrate the ability to rapidly reduce glucose levels in diabetic rodent models, replicating the timescale and therapeutic effect of subcutaneous insulin injection or infusion. This advance provides a potential path toward achieving insulin independence with islet macroencapsulation. Article Highlights: Numerous islet macroencapsulation techniques use diffusion to transport insulin across an immunoisolating barrier. Despite some devices reaching clinical trials, none have achieved physiological glucose control or insulin independence. Empirical evidence shows that high-density islet macroencapsulation methods cannot achieve on-demand insulin delivery and glucose regulation with diffusion alone. An additional driving force is needed. Appling a subminute pressure at physiological levels can achieve on-demand insulin delivery from macroencapsulated islets and glucose regulation. Incorporating pressure-based enhancements in macroencapsulation systems could lead to successful clinical outcomes.