Purpose: The gut microbiota plays important roles in health and disease. We questioned whether the gut microbiota and related metabolites are altered in monoclonal gammopathies and evaluated their potential role in multiple myeloma and its response to treatment. Experimental Design: We used 16S rRNA sequencing to characterize and compare the gut microbiota of patients with monoclonal gammopathy of undetermined significance (n = 11), smoldering multiple myeloma (n = 9), newly diagnosed multiple myeloma (n = 11), relapsed/refractory multiple myeloma (n = 6), or with complete remission (n = 9). Short-chain fatty acids (SCFA) were quantified in serum and tested in cell lines. Relevant metabolites were validated in a second cohort of 62 patients. Results: Significant differences in alpha- and beta diversity were present across the groups and both were lower in patients with relapse/refractory disease and higher in patients with complete remission after treatment. Differences were found in the abundance of several microbiota taxa across disease progression and in response to treatment. Bacteria involved in SCFA production, including Prevotella, Blautia, Weissella, and Agathobacter, were more represented in the premalignant or complete remission samples, and patients with higher levels of Agathobacter showed better overall survival. Serum levels of butyrate and propionate decreased across disease progression and butyrate was positively associated with a better response. Both metabolites had antiproliferative effects in multiple myeloma cell lines. Conclusions: We demonstrate that SCFAs metabolites and the gut microbiota associated with their production might have beneficial effects in disease evolution and response to treatment, underscoring its therapeutic potential and value as a predictor.

Among the different mechanisms involved in oxidative stress, protein carbonylation and lipid peroxidation are both important modifications associated with the pathogenesis of several diseases, including cancer. Hematopoietic cells are particularly vulnerable to oxidative damage, as the excessive production of reactive oxygen species and associated lipid peroxidation suppress self-renewal and induce DNA damage and genomic instability, which can trigger malignancy. A richer understanding of the clinical effects of oxidative stress might improve the prognosis of these diseases and inform therapeutic strategies. The most common protein carbonylation and lipid peroxidation compounds, including hydroxynonenal, malondialdehyde, and advanced oxidation protein products, have been investigated for their potential effect on hematopoietic cells in several studies. In this review, we focus on the most important protein carbonylation and lipid peroxidation biomarkers in hematological malignancies, their role in disease development, and potential treatment implications.

FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloid disorders that are challenging with regard to diagnosis and clinical management. To study the similarities and differences between these disorders, we undertook a multicenter international study of one of the largest case series (CNL, n = 24; aCML, n = 37 cases, respectively), focusing on the clinical and mutational profiles (n = 53 with molecular data) of these diseases. We found no differences in clinical presentations or outcomes of both entities. As previously described, both CNL and aCML share a complex mutational profile with mutations in genes involved in epigenetic regulation, splicing, and signaling pathways. Apart from CSF3R, only EZH2 and TET2 were differentially mutated between them. The molecular profiles support the notion of CNL and aCML being a continuum of the same disease that may fit best within the myelodysplastic/myeloproliferative neoplasms. We identified 4 high-risk mutated genes, specifically CEBPA (β = 2.26, hazard ratio [HR] = 9.54, P = .003), EZH2 (β = 1.12, HR = 3.062, P = .009), NRAS (β = 1.29, HR = 3.63, P = .048), and U2AF1 (β = 1.75, HR = 5.74, P = .013) using multivariate analysis. Our findings underscore the relevance of molecular-risk classification in CNL/aCML as well as the importance of CSF3R mutations in these diseases.

In cases of treatment failure in acute myeloid leukemia (AML), the utility of mutational profiling in primary refractoriness and relapse is not established. We undertook a perspective study using next-generation sequencing (NGS) of clinical follow-up samples (n=91) from 23 patients with AML with therapeutic failure to cytarabine plus idarubicin or fludarabine. Cases of primary refractoriness to treatment were associated with a lower number of DNA variants at diagnosis than cases of relapse (median 1.67 and 3.21, respectively, p=0.029). The most frequently affected pathways in patients with primary refractoriness were signaling, transcription and tumor suppression, whereas methylation and splicing pathways were mainly implicated in relapsed patients. New therapeutic targets, either by an approved drug or within clinical trials, were not identified in any of the cases of refractoriness (0/10); however, 8 potential new targets were found in 5 relapsed patients (5/13) (p=0.027): 1 IDH2, 3 SF3B1, 2 KRAS, 1 KIT and 1 JAK2. Sixty-five percent of all variants detected at diagnosis were not detected at complete response (CR). Specifically, 100% of variants in EZH2, RUNX1, VHL, FLT3, ETV6, U2AF1, PHF6 and SF3B1 disappeared at CR, indicating their potential use as markers to evaluate minimal residual disease (MRD) for follow-up of AML. Molecular follow-up using a custom NGS myeloid panel of 32 genes in the post-treatment evaluation of AML can help in the stratification of prognostic risk, the selection of MRD markers to monitor the response to treatment and guide post-remission strategies targeting AML, and the selection of new drugs for leukemia relapse.

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.