The proteasome is a multi-subunit complex responsible for intracellular protein degradation, while only 3 subunits harbor proteolytic activity, the. β1, β2, and β5 subunits. Carfilzomib (CFZ), a second-generation proteasome inhibitor (PI), induces cell death in multiple myeloma (MM) by selective and irreversible β5 inhibition. Currently, the optimal CFZ dosing is still controversial, with the approved dosage ranging from 20 to 70mg/m2 in different regimens. Moreover, it remains to be explored whether high-dose CFZ can achieve superior anti-MM efficacy over low-dose and recapture response in relapsed/refractory (RR) MM patients progressing under low-dose CFZ. To address these issues, we analyzed the clinical data and the inhibition profiles of proteolytic proteasome subunits β1, β2, and β5 of RRMM patients treated with different CFZ doses. We prospectively collected clinical data and peripheral blood mononuclear cells (PBMC) of 103 patients with RRMM before and 3 hours after CFZ. PBMC were lysed and labelled for the activity of individual proteasome subunits using activity based proteasome probes and the proteasome subunits were separated using SDS-PAGE. The activity of constitutive and immunoproteasome β1, β2 and β5 subunits was evaluated by densitometry analysis. We then investigated the clinical data of 114 patients treated with CFZ combinations. Overall, 23, 27, 38, and 15 patients received 20, 27, 36, and 56 mg/m2 of CFZ, respectively. β5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas β2 and β1 were co-inhibited only by 36 and 56 mg/m2, respectively. Co-inhibition of β2 (P=0.0001) and β1 activity (P=0.0005) differed significantly between high-dose (36 or 56 mg/m2) and low-dose (20 or 27 mg/m2) CFZ. Subsequently, high-dose CFZ showed significantly more effective proteasome inhibition than low-dose drug in vivo (P=0.0003). We then investigated the clinical data of 114 MM patients treated with CFZ combinations Kd, KRD, and D-Kd. In the entire group, high-dose CFZ demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose. In the subgroup analysis of Kd, high-dose CFZ likewise showed improved PFS over low-dose (P=0.0006). In patients treated with KRD, PFS was significantly longer in patients who had received high-dose CFZ than low-dose (P=0.02), while lenalidomide dose did not affect PFS in our cohort. In light of this finding, we escalated the dose of CFZ to ≥ 36 mg/m2 in 16 patients who progressed during low-dose CFZ-containing therapies, and the doses of agents other than CFZ in the combination regimens remained the same. High-dose CFZ recaptured response (≥ partial remission) in 9 (56%) patients with a median PFS of 4.4 months. Here, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose CFZ (≥ 36 mg/m2) differs from that of low-dose CFZ by co-inhibition of β2 and β1 proteasome subunits and, consequently, high-dose CFZ achieves a superior anti-MM effect than low-dose and recaptures response in RRMM being resistant to low-dose CFZ.

The addition of monoclonal antibodies daratumumab, elotuzumab and isatuximab to the treatment of patients with multiple myeloma significantly improved the outcome and prolonged survival. Unfortunately, although many patients benefit, depth and duration of response are a problem. In order to improve efficacy of antibody-based immunotherapy, we aimed to combine CD38-directed antibodies daratumumab and isatuximab as well as SLAMF7-targeting elotuzumab with a CD47 blocking antibody to enhance phagocytosis of myeloma cells. Antibody-dependent cellular phagocytosis (ADCP) of malignant plasma cells is described to be one important mode of action of daratumumab, isatuximab and elotuzumab, respectively. Of note, CD47 is highly expressed on myeloma cells and allows evading immune recognition by myeloid cells, i.e. monocytes, macrophages and neutrophils. Binding of CD47 to SIRPα expressed on myeloid cells provides a strong ‘don't eat me’ signal and diminishes phagocytosis of tumor cells. Blocking the CD47-SIRPα axis, by a monoclonal antibody against CD47 or a SIRPα-Fc fusion protein can restore recognition of tumor cells by macrophages and enhance phagocytosis. In patients with Non-Hodgkin's lymphoma the combination of CD20 antibody rituximab with CD47 antibody magrolimab was clinically successful (Advani et al., NEJM 379:1711, 2018).

Proteasome inhibitor (PI) carfilzomib (CFZ) has activity superior to bortezomib (BTZ) and is increasingly incorporated in multiple myeloma (MM) frontline therapy and relapsed settings. Most MM patients ultimately experience PI-refractory disease, an unmet medical need with poorly understood biology and dismal outcome. Pharmacologic targeting of ABCB1 improved patient outcomes, including MM, but suffered from adverse drug effects and insufficient plasma concentrations. Proteomics analysis identified ABCB1 overexpression as the most significant change in CFZ-resistant MM cells. We addressed the functional role of ABCB1 overexpression in MM and observed significantly upregulated ABCB1 in peripheral blood malignant plasma cells (PCs) vs untreated patients' bone marrow PC. ABCB1 overexpression reduces the proteasome-inhibiting activity of CFZ due to drug efflux, in contrast to BTZ. Likewise, the cytotoxicity of established anti-MM drugs was significantly reduced in ABCB1-expressing MM cells. In search for potential drugs targeting ABCB1 in clinical trials, we identified the HIV protease inhibitors nelfinavir (NFV) and lopinavir (LPV) as potent functional modulators of ABCB1-mediated drug export, most likely via modulation of mitochondria permeability transition pore. NFV and LPV restored CFZ activity at therapeutically relevant drug levels and thus represent ready-to-use drugs to be tested in clinical trials to target ABCB1 and to re-sensitize PC to established myeloma drugs, in particular CFZ.

The first-in-class alkylating HDAC inhibitor EDO-S101 is highly synergistic with proteasome inhibition against multiple myeloma through activation of multiple pathways

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by degeneration of the lower motor neurons and progressive muscle weakness leading to paralysis and premature death in the most severe cases. Recently, we developed an efficient gene therapy approach for this disease. In particular, we rescued the severe SMA mouse model, SMNΔ7, using one single intravenous (IV) injection of a self-complementary adeno-associated serotype 9 viral vector (scAAV9) encoding the SMN1 gene under the control of an ubiquitous promoter (PGK). We also showed that intracerebroventricular (ICV) delivery of the same vector outperforms IV administration route for SMA gene therapy. This higher therapeutic effect was mediated by transduction of motor neurons and peripheral organs, including heart and liver. To determine the contribution of neuronal SMN expression in SMA mouse rescue, we produced an AAV vector encoding SMN driven by the neuron-specific promoter Synapsin (AAV9-SYN-SMN). We compared the therapeutic effect of the ICV injected AAV9-SYN-SMN to the ICV or IV delivered AAV9-PGK-SMN, in neonatal SMNΔ7 mice. Interestingly, injection of the AAV9-SYN-SMN, at 4,5x1010 vg/mice, did not significantly extended SMA mice survival. Since the transcriptional strength of the SYN promoter could be weaker than that of PGK, we injected a second group of mice with a higher dose (1.2x1011 vg/mice) of AAV9-SYN-SMN. The median survival of ICV AAV9-SYN-SMN injected mice remained significantly lower than AAV9-PGK-SMN injected mice (42 days vs 105 or 173 days for IV or ICV AAV9-PGK-SMN injected mice, respectively), although 70 to 90% of MNs expressed SMN. All but one AAV9-SYN-SMN injected mice died before the age of 70 days, and displayed severe necrosis of the tail and limb extremities. Together, these results demonstrate that SMN expression restricted to the central nervous system it is not sufficient to rescue SMA mice. This study evidences the crucial role of SMN in peripheral organs.

Adaptive resistance of myeloma to proteasome inhibition represents a clinical challenge, whose biology is poorly understood. Proteasome mutations were implicated as underlying mechanism, while an alternative hypothesis based on low activation status of the unfolded protein response was recently suggested (IRE1/XBP1-low model). We generated bortezomib- and carfilzomib-adapted, highly resistant multiple myeloma cell clones (AMO-BTZ, AMO-CFZ), which we analyzed in a combined quantitative and functional proteomic approach. We demonstrate that proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition, irrespective of a proteasome mutation, and uniformly show an 'IRE1/XBP1-low' signature. Adaptation of myeloma cells to proteasome inhibitors involved quantitative changes in >600 protein species with similar patterns in AMO-BTZ and AMO-CFZ cells: proteins involved in metabolic regulation, redox homeostasis, and protein folding and destruction were upregulated, while apoptosis and transcription/translation were downregulated. The quantitatively most upregulated protein in AMO-CFZ cells was the multidrug resistance protein (MDR1) protein ABCB1, and carfilzomib resistance could be overcome by MDR1 inhibition. We propose a model where proteasome inhibitor-adapted myeloma cells tolerate subtotal proteasome inhibition owing to metabolic adaptations that favor the generation of reducing equivalents, such as NADPH, which is supported by oxidative glycolysis. Proteasome inhibitor resistance may thus be targeted by manipulating the energy and redox metabolism.

Bortezomib (BTZ)-resistant multiple myeloma (MM) cells generated in vitro by low concentration drug exposure (BTZ-adapted cells) are a widely used model for proteasome inhibitor (PI) resistance, and proteasome 5 active site mutations were implicated as resistance mechanism. BTZ-resistant, patient-derived MM cells lack such mutations. Their BTZ resistance is putatively mediated by downregulation of IRE-1/sXBP-1 pathway. We investigated if the in vitro model of PI-adapted MM is consistent with these hallmarks of primary MM cells, and compared BTZ- or CFZ-adapted MM cells regarding their patterns of adaptive changes and sensitivity against next generation PI (ixazomib – IXA, delanzomib – DLZ, oprozomib – OPR, the 2 selective inhibitor LU-102 and two novel 5/2-targeted PI).

We recently showed that a single intravenous injection of an optimized SMN-encoding scAAV9 (scAAV9-SMNopti) rescued a Spinal Muscular Atrophy (SMA) mouse model. Intramuscular (IM) injection of AAV1–AAV8 has been reported to mediate a moderate spinal cord transduction through axonal retrograde transport. Given the remarkable effectiveness of scAAV9 for motor neurons (MNs) gene transfer, we analyzed whether IM injection of this vector was efficacious to target MNs throughout the entire spinal cord. A single injection of scAAV9-GFP was performed into the right gastrocnemius of adult mice (n = 16) and GFP expression was analyzed 4 week later by immunofluorescence analysis on transversal spinal cord sections. The analysis of GFP expression revealed an efficient transduction of MNs throughout the lumbar spinal cord. Unexpectedly, cells in the thoracic and cervical spinal cord (which do not innervate the gastrocnemius) were also highly transduced, with the highest expression observed in the cervical segment. We therefore investigated the interest of this approach for SMA gene therapy in a SMNdelta7 mouse model. Neonatal muscle delivery of increasing doses of scAAV9-SMNopti in this model (n = 32), allowed restoration of survival in 100% of the treated mice. At the highest dose, the treatment increased life expectancy from 163 to over 227 days, with to date a mean survival of 188 ± 20 days versus 13.7 ± 0.6 days for untreated SMA mice. The intramuscular treatment also preserved motor activity, consistent with a protective effect on motor units. This study demonstrates, for the first time, that a single intramuscular scAAV9 injection into the hind limb of adult mice mediated MNs transduction in the whole spinal cord, and that this strategy was efficient to rescue a mouse model of severe SMA.