Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59s hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(βTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.

Background

Lower-grade IDH mutant glioma patients frequently undergo malignant transformation (MT), with apparent worse prognosis. Many studies examine MT in mixed IDH status cohorts and define MT using imaging, not histopathology. Our study examines the timing, predictors, and prognostic implications of pathologically determined MT in a large, exclusively IDH mutant cohort.

Methods

We identified 193 IDH mutant lower-grade glioma patients at UCLA who received multiple surgeries. We examined the outcomes of pathologically determined MT patients.

Results

Time to MT is longer in grade 2 oligodendroglioma (G2 Oligo) than in grade 2 astrocytoma (G2 Astro) (HR = 0.46, P = .0007). The grade 3 astrocytoma (G3 Astro) to grade 4 astrocytoma (G4 Astro) interval is shorter in stepwise MT (G2 to G3 to G4 Astro) patients than in initial G3 Astro patients (P = .03). Novel contrast enhancement had 65% positive predictivity, 67% negative predictivity, 75% sensitivity, and 55% specificity in indicating pathologically defined MT. In G2 Astro, initial gross total resection delayed MT (HR = 0.50, P = .02) and predicted better overall survival (OS) (HR = 0.34, P = .009). In G2 Oligo, spontaneous MT occurred earlier than treated MT (HR = 11.43, P = .0002), but treatment did not predict improved OS (P = .8). MT patients (n = 126) exhibited worse OS than non-MT patients (n = 67) in All (HR = 2.54, P = .0009) and G2 Astro (HR = 4.26, P = .02).

Conclusion

Our study expands the understanding of MT to improve IDH mutant lower-grade glioma management.

Background

Tumor surveillance of isocitrate dehydrogenase (IDH) mutant gliomas is accomplished via serial contrast MRI. When new contrast enhancement (CEnew) is detected during postsurgical surveillance, clinicians must assess whether CEnew indicates pseudoprogression (PsP) or tumor progression (TP). PsP has been better studied in IDH wild-type glioblastoma but has not been well characterized in IDH mutant gliomas. We conducted a retrospective study evaluating the incidence, predictors, natural history, and survival of PsP patients in a large cohort of IDH mutant glioma patients treated at a single institution.

Methods

We identified 587 IDH mutant glioma patients treated at UCLA. We directly inspected MRI images and radiology reports to identify CEnew and categorized CEnew into TP or PsP using MRI or histopathology.

Results

Fifty-six percent of patients developed CEnew (326/587); of these, 92/326 patients (28% of CEnew; 16% of all) developed PsP and 179/326 (55%) developed TP. All PsP patients had prior radiation, chemotherapy, or chemoradiotherapy. PsP was associated with longer overall survival (OS) versus TP patients and similar OS versus no CEnew. PsP differs from TP based on earlier time of onset (median 5.8 vs 17.4 months from treatment, P < .0001) and MRI features that include punctate enhancement and enhancement location.

Conclusion

PsP patients represented 28% of CEnew patients and 16% of all patients; PsP patients demonstrated superior outcomes to TP patients, and equivalent survival to patients without CEnew. PsP persists for <1 year, occurs after treatment, and differs from TP based on time of onset and radiographic features. Poor outcomes after CEnew are driven by TP.

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(βTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.