Venetoclax resistance leads to broad resistance to standard-of-care anti-MM agents, but not to immunotherapies.

Venetoclax is the first example of personalized medicine for multiple myeloma (MM), with meaningful clinical activity as a monotherapy and in combination in myeloma patients harboring the t(11:14) translocation. However, despite the high response rates and prolonged PFS, a significant proportion of patients eventually relapse. Here, we aimed to study adaptive molecular responses after the acquisition of venetoclax resistance in sensitive t(11:14) MM cell models. We therefore generated single-cell venetoclax-resistant t(11:14) MM cell lines and investigated the mechanisms contributing to resistance as well as the cells' sensitivity to other treatments. Our data suggests that acquired resistance to venetoclax is characterized by reduced mitochondrial priming and changes in BCL-2 family proteins' expression in MM cells, conferring broad resistance to standard-of-care anti-myeloma drugs. However, our results show that the resistant cells are still sensitive to immunotherapeutic treatments, highlighting the need to consider appropriate sequencing of these treatments following venetoclax-based regimens.

Bone marrow stromal cells induce chromatin remodeling in multiple myeloma cells leading to transcriptional changes.

The natural history of multiple myeloma is characterized by its localization to the bone marrow and its interaction with bone marrow stromal cells. The bone marrow stromal cells provide growth and survival signals, thereby promoting the development of drug resistance. Here, we show that the interaction between bone marrow stromal cells and myeloma cells (using human cell lines) induces chromatin remodeling of cis-regulatory elements and is associated with changes in the expression of genes involved in the cell migration and cytokine signaling. The expression of genes involved in these stromal interactions are observed in extramedullary disease in patients with myeloma and provides the rationale for survival of myeloma cells outside of the bone marrow microenvironment. Expression of these stromal interaction genes is also observed in a subset of patients with newly diagnosed myeloma and are akin to the transcriptional program of extramedullary disease. The presence of such adverse stromal interactions in newly diagnosed myeloma is associated with accelerated disease dissemination, predicts the early development of therapeutic resistance, and is of independent prognostic significance. These stromal cell induced transcriptomic and epigenomic changes both predict long-term outcomes and identify therapeutic targets in the tumor microenvironment for the development of novel therapeutic approaches.

Loss of GABARAP mediates resistance to immunogenic chemotherapy in multiple myeloma.

ABSTRACT: Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.

Round Table Discussion on Optimal Clinical Trial Design in Precursor Multiple Myeloma.

SUMMARY: While the current approach to precursor hematologic conditions is to "watch and wait," this may change with the development of therapies that are safe and extend survival or delay the onset of symptomatic disease. The goal of future therapies in precursor hematologic conditions is to improve survival and prevent or delay the development of symptomatic disease while maximizing safety. Clinical trial considerations in this field include identifying an appropriate at-risk population, safety assessments, dose selection, primary and secondary trial endpoints including surrogate endpoints, control arms, and quality-of-life metrics, all of which may enable more precise benefit-risk assessment.

DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

ABL1 kinase plays an important role in spontaneous and chemotherapy-induced genomic instability in multiple myeloma.

ABSTRACT: Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination (HR). Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either short hairpin RNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 reduces the HR activity and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan's efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, reduces genomic instability in live cell fraction, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in patients with MM.

Differentiation of BCMA-specific induced pluripotent stem cells into rejuvenated CD8αß+ T cells targeting multiple myeloma.

ABSTRACT: A major hurdle in adoptive T-cell therapy is cell exhaustion and failure to maintain antitumor responses. Here, we introduce an induced pluripotent stem cell (iPSC) strategy for reprogramming and revitalizing precursor exhausted B-cell maturation antigen (BCMA)-specific T cells to effectively target multiple myeloma (MM). Heteroclitic BCMA72-80 (YLMFLLRKI)-specific CD8+ memory cytotoxic T lymphocytes (CTL) were epigenetically reprogrammed to a pluripotent state, developed into hematopoietic progenitor cells (CD34+ CD43+/CD14- CD235a-), differentiated into the T-cell lineage and evaluated for their polyfunctional activities against MM. The final T-cell products demonstrated (1) mature CD8αß+ memory phenotype, (2) high expression of activation or costimulatory molecules (CD38, CD28, and 41BB), (3) no expression of immune checkpoint and senescence markers (CTLA4, PD1, LAG3, and TIM3; CD57), and (4) robust proliferation and polyfunctional immune responses to MM. The BCMA-specific iPSC-T cells possessed a single T-cell receptor clonotype with cognate BCMA peptide recognition and specificity for targeting MM. RNA sequencing analyses revealed distinct genome-wide shifts and a distinctive transcriptional profile in selected iPSC clones, which can develop CD8αß+ memory T cells. This includes a repertoire of gene regulators promoting T-cell lineage development, memory CTL activation, and immune response regulation (LCK, IL7R, 4-1BB, TRAIL, GZMB, FOXF1, and ITGA1). This study highlights the potential application of iPSC technology to an adaptive T-cell therapy protocol and identifies specific transcriptional patterns that could serve as a biomarker for selection of suitable iPSC clones for the successful development of antigen-specific CD8αß+ memory T cells to improve the outcome in patients with MM.

Acute Lymphoblastic Leukemia with Myeloid Mutations Is a High-Risk Disease Associated with Clonal Hematopoiesis.

Myeloid neoplasms arise from preexisting clonal hematopoiesis (CH); however, the role of CH in the pathogenesis of acute lymphoblastic leukemia (ALL) is unknown. We found that 18% of adult ALL cases harbored TP53, and 16% had myeloid CH-associated gene mutations. ALL with myeloid mutations (MyM) had distinct genetic and clinical characteristics, associated with inferior survival. By using single-cell proteogenomic analysis, we demonstrated that myeloid mutations were present years before the diagnosis of ALL, and a subset of these clones expanded over time to manifest as dominant clones in ALL. Single-cell RNA sequencing revealed upregulation of genes associated with cell survival and resistance to apoptosis in B-ALL with MyM, which responds better to newer immunotherapeutic approaches. These findings define ALL with MyM as a high-risk disease that can arise from antecedent CH and offer new mechanistic insights to develop better therapeutic and preventative strategies. SIGNIFICANCE: CH is a precursor lesion for lymphoblastic leukemogenesis. ALL with MyM has distinct genetic and clinical characteristics, associated with adverse survival outcomes after chemotherapy. CH can precede ALL years before diagnosis, and ALL with MyM is enriched with activated T cells that respond to immunotherapies such as blinatumomab. See related commentary by Iacobucci, p. 142.