Transcriptional Heterogeneity Overcomes Super-Enhancer Disrupting Drug Combinations in Multiple Myeloma.

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma.

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy.

BCMA-CD3-targeting bispecific antibodies (BsAb) are a recently developed immunotherapy class which shows potent tumor killing activity in multiple myeloma (MM). Here, we investigated a murine BCMA-CD3-targeting BsAb in the immunocompetent Vk*MYC and its IMiD-sensitive derivative Vk*MYChCRBN models of MM. The BCMA-CD3 BsAb was safe and efficacious in a subset of mice, but failed in those with high-tumor burden, consistent with clinical reports of BsAb in leukemia. The combination of BCMA-CD3 BsAb with pomalidomide expanded lytic T cells and improved activity even in IMiD resistant high-tumor burden cases. Yet, survival was only marginally extended due to acute toxicity and T cell exhaustion, which impaired T cell persistence. In contrast, the combination with cyclophosphamide was safe and allowed for a tempered pro-inflammatory response associated with long-lasting complete remission. Concurrent cytotoxic therapy with BsAb actually improved T cell persistence and function, offering a promising approach to patients with a large tumor burden.

Monosomic loss of MIR15A/MIR16-1 is a driver of multiple myeloma proliferation and disease progression.

The most common genetic abnormality in multiple myeloma (MM) is the deletion of chromosome 13, seen in almost half of newly diagnosed patients. Unlike chronic lymphocytic leukemia, where a recurrent minimally deleted region including MIR15A/MIR16-1 has been mapped, the deletions in MM predominantly involve the entire chromosome and no specific driver gene has been identified. Additional candidate loci include RB1 and DIS3, but while biallelic deletion of RB1 is associated with disease progression, DIS3 is a common essential gene and complete inactivation is not observed. The Vk*MYC transgenic mouse model of MM spontaneously acquires del(14), syntenic to human chromosome 13, and Rb1 complete inactivation, but not Dis3 mutations. Taking advantage of this model, we explored the role in MM initiation and progression of two candidate loci on chromosome 13: RB1 and MIR15A/MIR16-1. Monoallelic deletion of Mir15a/Mir16-1 but not Rb1 was sufficient to accelerate the development of monoclonal gammopathy in wildtype mice, and the progression of MM in Vk*MYC mice, resulting in increased expression of Mir15a/Mir16-1 target genes and plasma cell proliferation, which was similarly observed in patients with MM.

De Novo Mutations in EBF3 Cause a Neurodevelopmental Syndrome.

Early B cell factor 3 (EBF3) is an atypical transcription factor that is thought to influence the laminar formation of the cerebral cortex. Here, we report that de novo mutations in EBF3 cause a complex neurodevelopmental syndrome. The mutations were identified in two large-scale sequencing projects: the UK Deciphering Developmental Disorders (DDD) study and the Canadian Clinical Assessment of the Utility of Sequencing and Evaluation as a Service (CAUSES) study. The core phenotype includes moderate to severe intellectual disability, and many individuals exhibit cerebellar ataxia, subtle facial dysmorphism, strabismus, and vesicoureteric reflux, suggesting that EBF3 has a widespread developmental role. Pathogenic de novo variants identified in EBF3 include multiple loss-of-function and missense mutations. Structural modeling suggested that the missense mutations affect DNA binding. Functional analysis of mutant proteins with missense substitutions revealed reduced transcriptional activities and abilities to form heterodimers with wild-type EBF3. We conclude that EBF3, a transcription factor previously unknown to be associated with human disease, is important for brain and other organ development and warrants further investigation.

Visuomotor Correction is a Robust Contributor to Force Variability During Index Finger Abduction by Older Adults.

We examined aging-related differences in the contribution of visuomotor correction to force fluctuations during index finger abduction via the analysis of two datasets from similar subjects. Study (1) Young (N = 27, 23 ± 8 years) and older adults (N = 14, 72 ± 9 years) underwent assessment of maximum voluntary contraction force (MVC) and force steadiness during constant-force (CF) index finger abduction (2.5, 30, 65% MVC). For each trial, visual feedback of the force (VIS) was provided for 8-10 s and removed for 8-10 s (NOVIS). Visual gain of the force feedback at 2.5% MVC was high; 12- and 26-fold greater than the 30 and 65% MVC targets. Mean force, standard deviation (SD) of force, and coefficient of variation (CV) of force was calculated for detrended (<0.5 Hz drift removed) VIS and NOVIS data segments. Study (2) A similar group of 14 older adults performed discrete, randomly-ordered VIS or NOVIS trials at low target forces (1-3% MVC) and high visual gain. Study (1) For young adults the CV of force was similar between VIS and NOVIS for the 2.5% (4.8 vs. 4.3%), 30% (3.2 vs. 3.2%) and 65% (3.5 vs. 4.2%) target forces. In contrast, for older adults the CV of force was greater for VIS than NOVIS for 2.5% MVC (6.6 vs. 4.2%, p < 0.001), but not for the 30% (2.4 vs. 2.4%) and 65% (3.1 vs. 3.3%) target forces. At 2.5% MVC, the increase in CV of force for VIS compared with NOVIS was significantly greater (age × visual condition p = 0.008) for older than young adults. Study (2) Similarly, for older adults performing discrete, randomly ordered trials the CV of force was greater for VIS than NOVIS (6.04 vs. 3.81%, p = 0.01). When visual force feedback was a dominant source of information at low forces, normalized force variability was ~58% greater for older adults, but only 11% greater for young adults. The significant effect of visual feedback for older adults was not dependent on the order of presentation of visual conditions. The results indicate that impaired processing of visuomotor information underlies the greater motor variability observed in older adults during lab-based isometric contractions of a hand muscle.

Motor variability: within-subject correlations during separate and simultaneous contractions.

To determine the similarity of motor variability in proximal muscles, young and elderly adults performed steady elbow flexor (EF) and knee extensor (KE) contractions separately (SEP; at 2.5, 30, and 65% of maximum) and simultaneously (SIM; at 2.5 and 30% of maximum), with (VIS) and without (NVIS) visual feedback. Between-muscle correlations of fluctuation amplitude (SD, CV of force), time-based cross-correlations (CC), force power spectra, and frequency-based coherence (COH) values were computed from the concurrent force records. Correlations of fluctuation amplitude ranged from r = 0.34 to 0.86 (P < 0.05) across forces, SEP/SIM, and vision conditions, but were absent for 2.5% NVIS. The relatively low CC values for SIM (r = 0.22-0.33) were stronger for elderly than young adults. The vast majority of the power in the force fluctuations was <4 Hz for all records. Weak COH peaks were only observed <2 Hz for elderly and between 3 and 4 Hz for young, and COH was slightly stronger for elderly below 3 Hz for the 30% MVC target force. The correlations in force fluctuation amplitude suggest that the EF and KE motor neuron pools similarly transform the oscillating synaptic input and may influence each other. The cross-correlations suggest the remote motor neuron pools are influenced similarly in time by a common source of excitation, perhaps more coherently for elderly adults at low frequencies.

Aging, visuomotor correction, and force fluctuations in large muscles.

PURPOSE: To determine the contribution of visuomotor correction to increased force fluctuations in the elbow flexor and knee extensor muscles of elderly adults. METHODS: Young (N = 22, 23 +/- 3 yr) and elderly (N = 23, 74 +/- 7 yr) adults performed constant-force contractions at target forces of 2.5, 30, and 65% MVC. Visual feedback was provided (6-8 s) and then removed (6-8 s). After removal of drift (< 0.5 Hz) from the force, the standard deviation (SD) and coefficient of variation (CV) of force were calculated from vision and no-vision data. RESULTS: Maximal voluntary contraction (MVC) force was 19% lower for elbow flexors and 37% lower for knee extensors in elderly adults than in young adults. Overall, the CV of force was 27% greater in the vision condition compared with the no-vision condition. The CV of force for vision was greater for elderly adults than for young adults at the 2.5% MVC target force and lower at 30 and 65% MVC. For the 2.5% MVC target force, the decline in CV of force from vision to no vision was greater for elderly adults than for young adults. At 30 and 65% MVC, the decline was significant but similar for young and elderly adults. For elbow flexors, the change in power from vision to no vision was greater for 0- to 4-Hz (reduced power) and 8- to 12-Hz (increased power) frequencies for elderly adults compared with young adults. CONCLUSION: Visuomotor correction contributed to force fluctuations in large proximal muscles. The contribution was greater for healthy elderly adults at low forces. Visuomotor processes thus contributed to the age-related increase in force fluctuations.

Variability of quadriceps femoris motor neuron discharge and muscle force in human aging.

The purpose was to determine the contribution of visual feedback and the effect of aging on the variability of knee extensor (KE) muscle force and motor unit (MU) discharge. Single MUs were recorded during two types of isometric trials, (1) visual feedback provided (VIS) and then removed (NOVIS) during the trial (34 MUs from young, 32 from elderly), and (2) only NOVIS (66 MUs from young, 77 from elderly) during the trial. Recruitment threshold (RT) ranged from 0-37% MVC. Standard deviation (SD) and coefficient of variation (CV) of muscle force and MU interspike interval (ISI) was measured during steady contractions at target forces ranging from 0.3 to 54% MVC. Force drift (<0.5 Hz) was removed before analysis. VIS/NOVIS trials: the decrease in the CV of ISI from VIS to NOVIS was greater for MUs from elderly (12.5 +/- 4.1 to 9.94 +/- 2.6%) than young (10.6 +/- 3.3 to 10.3 +/- 2.8%, age group x vision interaction, P = 0.006). The change in CV of force from VIS to NOVIS was significantly greater for elderly (1.45 to 1.05%) than young (1.42 to 1.41%). NOVIS only trials: for all MUs, the average RT (6.6 +/- 7.7 % MVC), target force above RT (1.20 +/- 2.7% MVC), SD of ISI (0.012 +/- 0.005 s), and CV of ISI (11.1 +/- 3.3%) were similar for young and elderly MUs. The CV of force was similar between age groups for trials between 0 and 3% MVC (1.74 +/- 0.74%) and was greater for young subjects from 3 to 10% MVC (1.47 +/- 0.5 vs. 1.21 +/- 0.4%) and >10% MVC (1.44 +/- 0.6 vs. 1.01 +/- 0.3%). The CV of ISI was similar between age groups for MUs in 0-3, 3-10, and >10% bins of RT. Thus, the contribution of visuomotor correction to the variability of motor unit discharge and force is greater for elderly adults. The presence of visual feedback appears to be necessary to find greater discharge variability in motor units from the knee extensors of elderly adults.