Welcome to the MDS 2023 Congress Calendar

Abstract Body

MDS with deletion 5q was the first discrete MDS subgroup identified in formal nosology that was based on a genetic abnormality. While MDS with an isolated del(5q) may have distinct pathologic features and even disease manifestations, it also was subsequently discovered to have unique responsiveness to the immunomodulatory agent lenalidomide. Lenalidomide was found to exert its effects through inhibition of phosphatase activity in the common deleted region, which plays a key role in cell cycle regulation; and through a defect in ribosomal protein function via ubiquitination and degradation of CK1 alpha in patients with the deletion 5q cytogenetic abnormality. Approximately two-thirds of patients with lower-risk, transfusion-dependent MDS achieve transfusion independence lasting 8 weeks or longer to lenalidomide, a response rate that approximately doubles most other drugs used to treat lower-risk disease. The median duration of response – 2.2 years – also more than doubles typical response durations compared to other drugs, likely reflecting direct activity on the MDS clones, as does treatment -related thrombocytopenia, which predicts future erythroid response. For higher-risk MDS with del (5q), particularly in the setting of excess blasts, the karyotypic abnormality is rarely an isolated finding, and rarely amendable to lenalidomide monotherapy. Combination therapies that include lenalidomide do not appear to enhance responsiveness in higher-risk del(5q) patients, and lack of activity to lenalidomide-based therapy associated with acquisition of a TP53 mutation. Other treatment options for this MDS subgroup include erythropoiesis stimulating agents (which are often used prior to lenalidomide) in lower-risk patients, and hypomethylating agents (often used following lenalidomide) in higher-risk patients.

Abstract Body

My research program has developed a platform of functional approaches for analysis of primary cells from patients with a variety of hematologic malignancies, including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic disorder/MPN overlap (MDS/MPN), and AML that has transformed from MDS, MPN, or MDS/MPM. This platform aims to identify the druggable landscape across hematologic malignancies as well as match combination therapies with patient subsets in a precise manner. This pipeline is comprised of gene knockdown (e.g. siRNA, CRISPR/Cas) and small-molecule libraries to interrogate the genes and signaling pathways required by cancer cells, and these functional screens are complemented with next generation sequencing. Simultaneous application of these functional approaches with genomic data has accelerated our understanding of pathways that contribute to neoplasia. One example of this platform has been our Beat AML 1.0 platform in which we have assessed ex vivo drug sensitivity, whole exome sequencing, and RNA-sequencing for a cohort of nearly 1,000 AML patient samples, which provides a functional genomic landscape for this disease. We have also utilized genome-wide CRISPR screens with overlay of key therapeutic agents in cell line models, and coupling of these genome-wide screen data with the large patient sample dataset has illuminated a diversity of determinants of therapeutic response. In this lecture, I will provide an overview of these functional screening platforms, and I will illustrate strategies of data integration that have enabled rapid discovery and translation of new targeted therapy strategies. This will include discussion of novel biomarkers of drug response, including mutational drivers of drug response and strong correlation of tumor cell maturation state with a broad diversity of drugs and drug families. I will also discuss a machine learning-based approach for comprehensive analysis of the most robust determinants of clinical outcome, which has led to the discovery of a new and potentially targetable gene. This gene, PEAR1, exhibits higher expression across most poor prognostic subsets of AML, including elevated expression in MDS-transformed AML, and PEAR1 is strongly correlated with poor clinical outcome.

Abstract Body

Mutations in RNA splicing factors are the single most common class of genomic alterations in patients with myelodysplastic syndromes (MDS) and also occur in the setting of clonal hematopoiesis which predates MDS for many patients. Splicing factor mutations serve as attractive therapeutic targets as they frequently occur as early initiating events, are present in dominant clones, and are found in cancers with few effective treatment options. Spliceosomal mutations in SF3B1, SRSF2 and U2AF1 typically harbor mutually-exclusive heterozygous mutations and co-expression of these mutations is intolerable to cells. Furthermore, several studies have shown that splicing mutant cells are preferentially dependent on wild-type spliceosome function with deletion of the wild-type allele in splicing factor mutant cancer cells leading to cell death across different cancer subtypes with mutations in SF3B1, SRSF2, or U2AF1. These data highlight the potential vulnerability of splicing factor mutant cells to global perturbations in splicing catalysis and provide a therapeutic rationale for targeting splicing to trigger cell death.

This presentation will discuss several means to therapeutically target splicing factor mutant MDS. These include small molecules which bind to the SF3b component of the spliceosome, RBM39 degrading compounds, small molecule inhibitors targeting U2AF splicing interactions, and a number of enzymes which place post-translational modifications on RNA splicing factors. For this latter category we will specifically discuss enzymatic inhibitors of CLK and DYRK kinases as well as PRMT inhibitors.

Beyond chemicals, this presentation will also discuss a suite of synthetic RNA species which are specifically recognized by mutant splicing factors to drive selective gene expression. We have harnessed this approach to selectively eliminate splicing factor mutant cells but also to identify drugs and proteins required by the mutant spliceosome.

Finally, research from our laboratories has identified that the most common spliceosomal mutations alter RNA recognition in a sequence-specific manner to cause widespread mis-splicing. This widespread production of mis-spliced mRNAs, many of which encode novel peptides, could result in high levels of neoantigen production. Indeed, our preliminary studies have identified such putative neoantigens that are generated via mis-splicing arising from oncogenic spliceosomal mutations, presented by MHC class I, and capable of stimulating a cytotoxic T cell response. This work will also be discussed.

Background And Aims

Splicing factors (SFs) are among the most frequent mutational targets in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). CLK family of kinases phosphorylates a number of SFs containing RS domains, which is essential for RNA splicing and as such, can potentially be used to target SF-mutated MDS/AML.

Methods

We developed a highly potent and selective CLK inhibitor, CTX-712 and evaluated its anti-leukemic activities both in vitro and in vivo.

Results

CTX-712 suppressed phosphorylation of several RS domain-containing SFs including SRSF2/3/4/6. Consistent with the biological function of SR proteins in exon recognition, RNA-seq analysis revealed that CTX-712 induced global aberrant splicing changes in MV-4-11 and K562 cells, which largely consisted of exon skipping. Isogenic cell lines harboring SF mutations showed a higher sensitivity to CTX-712 than un-mutated cells. We next evaluated effects of CTX-712 on primary and established AML cells, as well as MDS/AML-derived xenografts (PDX). Unexpectedly, regardless of their SF-mutation status, a wide variety of primary and established myeloid leukemia cells and PDX models exhibited high sensitivity to CTX-712. Importantly, the effect of CTX-712 positively correlated with the degree of altered splicing in cassette exon events induced by the drug in AML cell lines, primary AML cells and PDX models, suggesting that the anti-leukemic effects of CTX-712 are mediated by inhibition of RNA splicing.

Conclusions

CTX-712 is a potent and specific inhibitor of CLK kinases showing a broad activity to a wide variety of MDS and AML, highlighting a rationale for further investigation of CTX-712 in MDS/AML.

Background And Aims

Myelodysplastic syndromes (MDS) represent hematological malignancies characterized by defective differentiation of hematopoietic stem and progenitor cells (HSPCs). The aim of this study was to perform a detailed transcriptional characterization of HSPCs in order to unravel novel mechanisms driving myeloid differentiation blockage in MDS.

Methods

We performed MARS-sequencing on FACS-sorted HSPCs (HSCs, CMPs, GMPs, MEPs) from healthy individuals (n=12) and untreated MDS patients with multi-lineage dysplasia (n=18). We developed a computational model to identify genes whose expression dynamics are altered in MDS.

Results

Our model identified 579 and 711 disrupted genes along the monocytic/granulocytic (HSC-CMP-GMP) and the megakaryocytic/erythrocytic (HSC-CMP-MEP) lineages, respectively. The vast majority of these genes showed altered trajectories with either higher or lower expression than expected across differentiation. Ontology studies on negatively altered genes suggested their participation in myeloid cells’ differentiation and functionality, including neutrophil activation/degranulation and gas transport. Further, the application of TraRe, a Gene-Regulatory-Network inference method, uncovered transcription factors that could be globally driving such transcriptomic dysregulations in MDS. Among them, ZNF350 and ZMAT2 were associated with key disrupted genes, and intriguingly, their inhibition by CRISPR-Cas9 triggered a higher differentiation potential in MDS cell lines, thus representing potential therapeutic targets for reverting differentiation blockage in MDS.

Conclusions

These findings offer a new approach in the study of MDS pathogenesis and shed light into novel drivers of aberrant hematopoiesis not described to date.

Acknowledgements: Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program. ISCIII co-financed by ERDF (PI17/00701, PI20/01308, PI 20/00531).