Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.

Complete inhibition of a polyol nucleation by a micromolar biopolymer additive.

Preventing spontaneous crystallization of supersaturated solutions by additives is of critical interest to successful process design and implementation, with numerous applications in chemical, pharmaceutical, medical, pigment, and food industries, but challenges remain in laboratory and industry settings and fundamental understanding is lacking. When copresented with antifreeze proteins (AFPs), otherwise spontaneously crystallizing osmolytes are maintained at high supersaturations for months in over-wintering organisms. Thus, we here explore the inhibition phenomenon by AFPs, using persistent crystallization of a common sugar alcohol, D-mannitol, as a case study. We report experimentally that DAFP1, an insect AFP, completely inhibits D-mannitol nucleation. Computer simulations reveal a new mechanism for crystallization inhibition where the population of the crystal-forming conformers are selectively bound and randomized in solution by hydrogen bonding to the protein surface. These results highlight the advantages of using natural polymers to address crystallization inhibition challenges and suggest new strategies in controlling the nucleation processes.