Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

Whole Genome Sequencing Identifies Microdeletions Affecting TET2 and RUNX1 with Clinical Impact in Myeloid Malignancies

Background: The current routine genetic work-up in hematological malignancies includes chromosome banding analysis (CBA) to detect complete or partial chromosomal deletions and fusions, and the identification of point mutations and small deletions or insertions by sequencing panels (max. length ~50 bp). Deletions of individual genes (e.g. IKZF1 in ALL) are only detected by specifically designed molecular tools. Therefore, those microdeletions might be overlooked by the current gold standard despite their clinical relevance. We established a bioinformatic pipeline to screen for microdeletions in whole genome sequencing (WGS) data of myeloid malignancies. Aim: (1) Screen for recurrent microdeletions in myeloid malignancies with a normal karyotype, and (2) characterize a patient specific profile of microdeletions in genes with known clinical and/or prognostic relevance. Patients and Methods: We analyzed 1356 cases (M/F: 778/578) of myeloid malignancies with a normal karyotype according to CBA (aCML: n=47; AML: n=251; CMML: n=165, mastocytosis: n=90; MDS: n=415, MDS/MPN-RS-T: n=69; MDS/MPN-U: n=42; MPN: n=250; PNH: n=27) using WGS. Median age was 71 [20-94] years. Amplification-free WGS was performed on the NovaSeq or HiSeq system with a median coverage of 103x (Illumina, San Diego, CA). Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and somatic copy number variant (CNV) discovery was performed with GATK (v 4.0.2.1), following best practice guidelines. Only gene overlapping CNV calls were considered for analysis (gene coordinates biomaRt (v 2.42.1), GRCh37 Ensembl). Results: On average, 38 genes per patient were partially or completely deleted and the size of the deletions ranged from 0.9 kb to 32 Mb (median 399 kb). The microdeletions affected a broad list of genes, but no gene was present in >5% of myeloid malignancies. As technical validation, we used 36 B-ALL samples (normal karyotype) and identified the known deletions of IKZF1 (42%); PAX5 (25%) and CDKN2A/CDKN2B (22%) with expected incidences. We focused on a patient-by-patient analysis of genes (n=47) with known clinical relevance in myeloid malignancies. We identified deleted genes in 46 out of 1356 patients (3.4%). In aCML 13% of patients had one of the above-mentioned genes deleted (6/47), in mastocytosis only 1% (1/90). The most frequently deleted genes were TET2 (20/1356, 1.5%) and RUNX1 (9/1356, 0.7%). Other deletions also affected transcription factors (e.g. GATA2) or epigenetic regulators (e.g. DNMT3A, figure 1). No deletion of splicing factors, RAS genes or cohesion complex regulators was observed. We found only two deletions of kinases, which are predominantly affected by activating mutations (both FLT3). Instead, the deletions in 41 patients involved genes with a known loss-of-function mutation profile in myeloid malignancies. This corresponds to 89% (41/46) of patients with microdeletions or 3% (41/1356) of all analyzed patients with myeloid malignancies. Microdeletions are thus another genetic element that can lead to loss of gene activity. Deletions and mutations are either alternative genetic mechanisms or co-operate as double hits to affect the same gene. We found additional mutations present in 18 of the 46 patients with microdeletions (39%, figure 1). The majority of these (n=14) involved TET2. TET2 mutations had a median variant allele frequency of 82% [9-100%] indicative of a mutation on the non-deleted allele. For the remaining genes (incl. RUNX1), deletions are predominantly an alternative genetic mechanism to mutations. For validation of WGS results we applied interphase FISH and identified 6/9 RUNX1 deletions. The remaining three microdeletions were only detectable by WGS and too small to be identified by FISH. Conclusions: (1) WGS data unrevealed a plethora of microdeletions, which can be an alternative genetic mechanism to mutations, but are not detected with today's standard diagnostic tools. (2) In the light of increasingly personalized therapy and diagnostics, all genetic mechanisms should be considered, which impact the function of clinically relevant genes. (3) Bioinformatic pipelines for WGS as a potential diagnostic tool in the near future should address microdeletions in genes with relevance for patients' diagnosis, prognosis and hopefully targeted treatment. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Novel Synthetic Lethal Targets for Myeloid Neoplasms with Loss of Chromosome 7

Loss of chr7 (-7) and partial deletions of its long arm (del7q) are observed in 10% of de novo myeloid neoplasms (MNs), 50% of therapy related MDS, up to 60% of post aplastic anemia MNs and occur frequently as evolution of congenital bone marrow failure syndromes (e.g., GATA2 and SAMD9L deficiency, FA). LOH of one or more chr7 genes has been considered the culprit in the pathogenesis of -7/del7q MNs. In addition to the loss of protective alleles, deletion resulting in haploinsufficiency (HI) of tumor suppressor genes (TSGs) in CDRs might be also a cause of leukemogenic drive behind -7/del7q. To date, albeit many candidate genes have been associated with -7/del7q, the search for genes responsible for clinical phenotype has failed to identify causative -7/del7q TSGs and their putative loss would be difficult to target. Irrespective of the important goal of clarification of leukemogenic effects of -7/del7q, loss of genes in CDRs may create a vulnerability phenotype, which could be exploited with synthetic lethal approaches. Such strategies would rely on the higher resistance of diploid vs -7/del7q cells, thus allowing for a therapeutic window. Here, we studied the molecular profile of 8160 MN patients (del7q: 1.7%; -7: 6%). EZH2 mutations were enriched in -7/del7q compared to chr7 diploid cases (3.8 vs 1.2%, P<.0001) also by absolute numbers heterozygous mutations were more numerous. We also detected somatic CUX1 mutations (1.7 vs 0.9%), SAMD9/SAMD9L (0.3 vs 0.1%), and LUC7L2 (0.3 vs 0.1%) in -7/del7q vs diploid. In -7/del7 cases somatic alterations were detected in BRAF (n=7), POT1 (n=3), PCLO (n=5) and PSMC2 (n=1) while no mutations in CUL1 and KMT2C were found. We then investigated the presence of driver mutations located on other chromosomes in -7/del7q. Del7q/-7 cases showed a lower frequency of TET2 and SF3B1 mutations vs diploid cases. In isolated/+1 del7q/-7 cases, TP53 mutations were significantly less frequent, but were increased in -7/del7q with complex karyotype (P<.0001). Higher frequencies of RAS genes, RUNX1 and ETV6 hits were also found. Del7q and TP53 mutations were founder lesions (dominant) in 38% and 54% of -7/del7q, while -7 was dominant in 63% of -7 cases. TP53 was the only mutation significantly associated with further worsening the already poor prognosis of -7/del7q cases (HR=1.629 P< .01). Germline alterations were more common in -7/del7q as compared to diploid cases (13 vs 5% P< .0001) of which most were FA or DNA repair gene variants also in other genes including (e.g., SAMD9L, 7%, DDX41, 3.7%). Having defined the genotype of -7/del7q, we set to identify genes which could be possible targets for the therapy chiefly synthetic lethality. Criteria for selection included: consistent HI in most of the patients, genes not affected by hemizygous LOF mutations and embryonic lethality in knockout (KO) configuration. Expression data of -7/del7q (n=86), diploid cases (n=1066) and healthy controls (n=84; MLL and BEAT AML to increase precision) were analyzed. Our algorithm included selection of genes with mRNA expression inversely correlating with copy number (deletion copy number). Out of 694 genes on chr7, 147 genes were deleted in all patients and 101 genes had more inconsistent HI levels. In total 35 genes showed significant negative correlation with -7/del7q ploidyincluding ACTR3B,AGK,ATP06V0E2,CUL1,FASTK,GALNT11,GSTK1, IMPDH1, PLXNA4, SLC37A3, ZNF277, KMT2C, NUP205, TMEM209, ZC3HC1 and GIMAP1/2/4/6, a cluster ofnucleotide binding proteins. Following adjustment to ploidy, HI was found for EZH2 (76% cases), CUX1 (76%), KMT2C (70%), LUC7L2 (60%), and SAMD9/9L (32%/50%) but also even more consistently in SSBP1 (88%), PSMC2 (86%), CUL1, ZNF398, and RHEB (all 84%) and TNPO3 (82%). Among those genes homozygous KO of Ezh2 and Cul1 lead to embryonic lethality, Gimap family deletion reduces normal hematopoiesis, Samd9l +/-and Samd9l-/- mice develop MDS and die after 1.5yrs and Cux1 knockdown causes an MDS like phenotype. Existing inhibitors are available for CUL1 (MLN4924), CUX1 (BER modulating agents) and EZH2 (EPZ6438, GSK343), but the presence of homozygous mutations (UPD7q) argues that EZH2 inhibition is unlikely to be successful. In conclusion, we showed a comprehensive molecular topography of -7/del7q and identified novel HI genes which could be targeted by novel or repurposed drugs. Ongoing drug screens for identified targets performed in cells with -7/del7q will be presented at the meeting. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.

Ras protein abundance correlates with Ras isoform mutation patterns in cancer.

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS>NRAS>>HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. However, they challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.

A személyre szabott terápia legújabb lehetőségei a molekuláris onkológiában

Összefoglaló. A molekuláris onkológia térnyerésével számos új lehetőség érhető el a daganatos betegek hatékonyabb kezelésére. Ilyen a klinikai vizsgálatokban alkalmazott, a valóban személyre szabott kezelést elősegítő génpanelelemzés, illetve a célzott kezelés szövettípustól független alkalmazása. A személyre szabott terápiák jelentős hányada valamelyik kinázt gátolja. Az összefoglalónkban bemutatjuk a RAS jelátviteli út sejten belüli komplex szabályozását, valamint ismertetjük az útvonal további farmakológiai szempontból kiaknázható célpontjait nemzetközi és saját eredményeink alapján. A kinázokat érintő gyakori mutációk ellenére számos daganattípusban nem áll rendelkezésre személyre szabott terápia. A hagyományos terápiával nem kezelhető agydaganatok példáján keresztül bemutatjuk a tirozin-kinázok várható jövőbeli terápiás jelentőségét. Summary. With the advent of molecular oncology, the identification of mutations in solid tumours is now clinically routine. The growing repertoire of targeted therapeutic agents has supported the rise of a new type of clinical trial in which the selection of the therapeutic agent is no longer restricted to a single option. Instead, a panel of genes is screened to identify the most suitable drug for each patient. Such trials have delivered objective response rates in 5–30% of patients. Most of the signal transduction pathways targeted by these agents involves RAS signaling. Somatic mutations in RAS genes are common in human tumours. Such mutations generally decrease the ability of RAS to hydrolyze GTP, maintaining the protein in a constitutively active GTP-bound form that drives uncontrolled cell proliferation. Recent emerging data suggest that RAS regulation is more complex than the scientific community has appreciated for decades. We discuss a novel type of RAS regulation that involves direct phosphorylation and dephosphorylation of RAS tyrosine residues. The discovery that pharmacological inhibition of the tyrosine phosphoprotein phosphatase SHP2 maintains mutant Ras in an inactive state suggests that SHP2 could be a novel drug target for the treatment of Ras-driven human cancers. In addition to RAS gene mutations, other common oncogenic events have also been identified, including mutation of EGFR (epidermal growth factor receptor) or BRAF (isoform B of rapidly accelerated fibrosarcoma). EGFR has tyrosine kinase activity while BRAF acts as a serine/threonine kinase. In some tumours, mutant forms of these kinases over-activate cell proliferation, leading to uncontrolled tumour cell growth; therefore, it seems rational to develop inhibitor molecules that target these hyper-active oncogenic kinases to reduce tumour cell burden in cancer patients. Fusion protein kinases activated via the RAS pathway represent target proteins with high clinical success rates. Recently approved TRK fusion protein kinase inhibitors have reached response rates in almost 80% of patients regardless of tumour type. Although these drugs can only be administered to patients whose tumours harbour a TRK fusion protein, such success stories pave the way for future development of agents that target similar genetic mutations. Glioblastoma multiforme, a relatively frequent, almost uniformly fatal brain tumour, has ubiquitous alterations in tyrosine-kinase signalling. Nevertheless, to this day, no tyrosine-kinase inhibitors have been approved for its treatment. We have ongoing research projects to uncover associations between initial gene expression levels in untreated glioblastoma samples and treatment-related survival, and we have identified overexpression of druggable tyrosine-kinase receptors in chemotherapy-resistant patients. Our approach will help to identify patients who might benefit from concurrent use of tyrosine kinase inhibitors and conventional cytotoxic therapies.

Progress on Ras/MAPK Signaling Research and Targeting in Blood and Solid Cancers

The mitogen-activated protein kinase (MAPK) pathway, consisting of the Ras-Raf-MEK-ERK signaling cascade, regulates genes that control cellular development, differentiation, proliferation, and apoptosis. Within the cascade, multiple isoforms of Ras and Raf each display differences in functionality, efficiency, and, critically, oncogenic potential. According to the NCI, over 30% of all human cancers are driven by Ras genes. This dysfunctional signaling is implicated in a wide variety of leukemias and solid tumors, both with and without viral etiology. Due to the strong evidence of Ras-Raf involvement in tumorigenesis, many have attempted to target the cascade to treat these malignancies. Decades of unsuccessful experimentation had deemed Ras undruggable, but recently, the approval of Sotorasib as the first ever KRas inhibitor represents a monumental breakthrough. This advancement is not without novel challenges. As a G12C mutant-specific drug, it also represents the issue of drug target specificity within Ras pathway; not only do many drugs only affect single mutational profiles, with few pan-inhibitor exceptions, tumor genetic heterogeneity may give rise to drug-resistant profiles. Furthermore, significant challenges in targeting downstream Raf, especially the BRaf isoform, lie in the paradoxical activation of wild-type BRaf by BRaf mutant inhibitors. This literature review will delineate the mechanisms of Ras signaling in the MAPK pathway and its possible oncogenic mutations, illustrate how specific mutations affect the pathogenesis of specific cancers, and compare available and in-development treatments targeting the Ras pathway.

Modern strategy for treatment of metastatic colorectal cancer as key to increasing life expectancy of patients with metastatic colorectal cancer without mutations in RAS genes

The review is devoted to the place of cetuximab in the treatment of metastatic colorectal cancer (mCRC) without mutations in the RAS (RAS wt) and BRAF (BRAF wt) genes, depending on the goals of therapy, as well as to the analysis of the inflence of various factors, including the localization of the primary tumor, on the effectiveness of treatment. Randomized clinical trials and meta-analyses conducted on their basis allow us to conclude that cetuximab in combination with an infusion doublet or triplet provides the maximum frequency of deep and early objective responses, regardless of the location of the primary tumor. The drug is superior in this parameter to both a single chemotherapy (CT) and a combination of CT with bevacizumab which is important in terms of achieving resectability in patients with potentially resectable metastases. For patients with left-sided localization of the primary tumor and RAS wt, cetuximab, prescribed in the 1st line, provides a reliable and clinically signifiant increase in life expectancy. Postponing the start of its use until 2–4 cycles of CT (until the result of a molecular genetic study is obtained) does not negatively affect the effectiveness of the 1st line of therapy for mCRC RAS wt, and with left-sided localization of the primary tumor, CT with delayed cetuximab exceeds the usage of CT with bevacizumab from the fist cycle for ORR, OS and PFS. The optimal duration of induction chemo-targeted therapy is 3–4 months (6–8 courses), after which it is advisable to switch to maintenance treatment with one cetuximab. The new mode of administration of cetuximab once every 2 weeks at a dosage of 500 mg/m 2 IV provides maximum convenience of its use.

Molecules linked to Ras signaling as therapeutic targets in cardiac pathologies

The Ras family of small Guanosine Triphosphate (GTP)-binding proteins (G proteins) represents one of the main components of intracellular signal transduction required for normal cardiac growth, but is also critically involved in the development of cardiac hypertrophy and heart failure. The present review provides an update on the role of the H-, K- and N-Ras genes and their related pathways in cardiac diseases. We focus on cardiac hypertrophy and heart failure, where Ras has been studied the most. We also review other cardiac diseases, like genetic disorders related to Ras. The scope of the review extends from fundamental concepts to therapeutic applications. Although the three Ras genes have a nearly identical primary structure, there are important functional differences between them: H-Ras mainly regulates cardiomyocyte size, whereas K-Ras regulates cardiomyocyte proliferation. N-Ras is the least studied in cardiac cells and is less associated to cardiac defects. Clinically, oncogenic H-Ras causes Costello syndrome and facio-cutaneous-skeletal syndromes with hypertrophic cardiomyopathy and arrhythmias. On the other hand, oncogenic K-Ras and alterations of other genes of the Ras-Mitogen-Activated Protein Kinase (MAPK) pathway, like Raf, cause Noonan syndrome and cardio-facio-cutaneous syndromes characterized by cardiac hypertrophy and septal defects. We further review the modulation by Ras of key signaling pathways in the cardiomyocyte, including: (i) the classical Ras-Raf-MAPK pathway, which leads to a more physiological form of cardiac hypertrophy; as well as other pathways associated with pathological cardiac hypertrophy, like (ii) The SAPK (stress activated protein kinase) pathways p38 and JNK; and (iii) The alternative pathway Raf-Calcineurin-Nuclear Factor of Activated T cells (NFAT). Genetic alterations of Ras isoforms or of genes in the Ras-MAPK pathway result in Ras-opathies, conditions frequently associated with cardiac hypertrophy or septal defects among other cardiac diseases. Several studies underline the potential role of H- and K-Ras as a hinge between physiological and pathological cardiac hypertrophy, and as potential therapeutic targets in cardiac hypertrophy and failure. Graphic abstract

Anthraquinones as Inhibitors of SOS RAS-GEF Activity

Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers.

Genetic polymorphisms in the renin-angiotensin system and cognitive decline in Parkinson’s disease

Background Renin-angiotensin system (RAS) influences the central nervous system not only through its peripheral impact—the brain possesses its own local RAS. Studies showed altered RAS components in Parkinson’s disease (PD) and their association with oxidative stress which may be linked to neurodegeneration and dementia. Moreover, the protective functions of RAS blockade antagonists against cognitive decline and dementia have been suggested. This study aimed to examine whether genetic variability in RAS genes correlates with cognitive decline in PD. Methods and results We genotyped single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT: rs699, rs4762), angiotensin II receptors (AGTR1: rs5186 and AGTR2: rs5194, rs1403543) genes, as well as insertion/deletion polymorphism in the angiotensin-converting enzyme (ACE I/D) gene in 256 PD patients, divided into three groups: without cognitive decline, with mild cognitive impairment and with PD dementia. We did not find any significant differences in the frequencies of the analysed polymorphisms in any of the groups. Conclusions Despite no direct correlation between the investigated polymorphisms in RAS genes and cognitive decline in PD, we believe the impact of those genotypes may be indirect, affecting RAS blockade treatment.