ALBERT

Description: Introduction and Methods To investigate the mechanisms by which microenvironmental signals induce treatment resistance in chronic lymphocytic leukemia (CLL) we conducted a high-throughput perturbation assay combining pairwise 15 clinically relevant drugs with 18 stimulations mimicking the tumor microenvironment. We combined this with multi-layer omics data using whole exome sequencing, genome-wide DNA-methylation profiles and RNA sequencing to investigate molecular determinants of microenvironmental drug resistance. In parallel we assessed the in-vivo relevance of investigated pathways in 100 CLL infiltrated- and 100 healthy lymph node (LN) biopsies by staining for stimulus specific downstream pathway components using immunohistochemistry (IHC). Results We initially clustered drugs and microenvironmental stimuli separately based on the similarity of their response profiles across all CLL samples. We found drug response profiles to be correlated closely for drugs targeting the same signaling-pathway, e.g. the B-cell receptor inhibitors Ibrutinib (BTK) and PRT062607 (Syk) (Pearson correlation coefficient: r=0.78, 95% CI: 0.72 - 0.83). In contrast, responses to microenvironmental stimuli were less correlated, indicating diverse effects are caused by the tumor microenvironment (e.g. Interleukin 4 (IL-4) vs. Interleukin 2, Pearson correlation coefficient: r=0.17, 95% CI: 0.03 - 0.31). Twelve of the 18 tested stimulations enhanced survival of CLL cells, whereas two reduced the viability of CLL cells in-vitro (comparisons yielded BH adjusted t-test p-values

Description: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse leukemogenic driver lesions. The genetic understanding of AML has resulted in major improvements in diagnosis, classification, prognostication, and outcome prediction. However, these insights have not yet translated into molecular mechanism-based therapies in the majority of cases, because AML cells rapidly escape attempts at therapeutic targeting such as small-molecule inhibition of FLT3 internal tandem duplication (FLT3-ITD) mutants, which occur in up to 30% of AML cases and confer a poor prognosis. To identify potential new targets for combinatorial treatment approaches, we performed a series of large-scale short hairpin RNA (shRNA) screens and observed that cell lines representing various AML subtypes were dependent on expression of the RET receptor tyrosine kinase (RTK), which has not previously been implicated in AML pathogenesis. Validation experiments demonstrated that depletion of RET by shRNA knockdown or CRISPR/Cas9-mediated knockout led to cell cycle arrest in the G0/G1 phase, increased apoptosis, and reduced clonogenic activity. RTK profiling using ELISA-based antibody arrays demonstrated that RET is highly phosphorylated in RET-dependent AML cell lines. Analysis of known RET ligand/co-receptor pairs (GDNF/GFRA1, NRTN/GFRA2, ARTN/GFRA3, PSPN/GFRA4) by quantitative real-time PCR and shRNA knockdown indicated that RET signaling is facilitated mainly through NTRN/GFRA2 or ARTN/GFRA3. Interrogation of various signaling pathways known to promote myeloid leukemogenesis showed that RET knockdown resulted in decreased phosphorylation of 4E-BP1 (T37/46), p70S6K (T389), S6RP (S240/244), and ULK1 (S758), pointing to mTORC1-mediated protein synthesis and/or suppression of autophagy as important effectors of RET signaling in AML cells. Based on recent data showing that FLT3-ITD mutants can be degraded by autophagy (Larrue et al. Blood 2016), we reasoned that the RET-mTORC1 signaling axis promotes AML through protection of FLT3-ITD mutants from autophagic degradation. Consistent with this hypothesis, genetic or pharmacologic (vandetanib, danusertib) inhibition of RET predominantly affected FLT3-dependent AML cell lines and were accompanied by upregulation of autophagy and destabilization of FLT3, as evidenced by p62 degradation, LC3B turnover, increased numbers of autophagic vacuoles, and decreased FLT3 protein levels. Furthermore, we observed accumulation of STAT5, a key FLT3-ITD downstream effector, upon pharmacologic autophagy inhibition in low RET-expressing AML cells, underlining the importance of RET-mediated suppression of autophagy for leukemogenic FLT3-ITD signaling. In line with the observations in AML cell lines, preliminary data from a murine bone marrow transplantation model show that Ret is required for AML development and propagation in vivo as we observed a significant survival advantage for mice transplanted with Ret knockdown cells compared with mice transplanted with control cells. Finally, genome-wide transcriptome analysis identified elevated RET mRNA levels in 35 of 260 (13.5%) primary human AML samples. Since there are no known RET copy number alterations or mutations of the RET coding region in AML patients and cell lines, we are currently investigating whether aberrant RET expression in AML can be attributed to perturbed epigenetic regulation. To this end, we are applying chromosome conformation capture combined with high-throughput sequencing (4C-seq) technology to systematically analyze interactions of the RET promoter region with enhancer sequences in high and low RET-expressing AML cell lines. Combined, our results indicate that in a proportion of AML, RET-mTORC1 signaling promotes cell viability and proliferation through suppression of autophagy, suggesting that targeting RET or, more broadly, depletion of critical leukemogenic drivers via induction of autophagy may provide a therapeutic opportunity in this subset of patients. Disclosures No relevant conflicts of interest to declare.

Description: Key Points The HAT complex member TRRAP is vital for maintaining high p53 levels by shielding it against the natural p53 degradation machinery. Acetylation-modifying complexes regulate p53 protein stability, which may provide a basis for therapeutic targeting of mutant p53.

Description: Background: Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. However, a systems-level understanding of how the microenvironment and the genetic and molecular alterations of the tumor interact with each other and contribute to drug resistance is lacking. Methods: To address this unmet need, we established an automated microscopy-based phenotyping platform that uses co-culture conditions mimicking the bone marrow environment. We cultured primary tumor cells from more than 100 leukemia patients (CLL, AML, MCL, T-PLL, HCL) with and without bone marrow stroma cell support in DMEM and 10% human serum and treated each condition with 57 drugs in 3 concentrations. After 72h of incubation, 22 000 images per patient were acquired and processed by our custom made image analysis pipeline. Our set-up allows us to increase sensitivity far beyond simple viability testing, as it reads out additional cell type specific features such as cell morphology, autophagy and cell-cell interactions. Results: Quality assessment revealed that in contrast to mono-culture conditions, assay plate edge effects can be avoided under stable stroma cell co-culture conditions. Correlation of replicated patient samples were comparable between mono- and co-cultures (R2〉0.75). In the absence of their native microenvironment, primary leukemia cells undergo spontaneous apoptosis ex-vivo. Viability at culture start was always 〉90% and dropped to a median of 51% (viability range: 17%-90%) after 72h in mono-cultures. Among CLL samples spontaneous apoptosis was not dependent on either IGHV mutation status or any major cytogenetic risk group. Bone marrow stroma cell co-culture conditions protected tumor cells from spontaneous apoptosis (p=8.2e-6, paired t-test). Patient samples with a high degree of spontaneous apoptosis benefited most from co-culture conditions (p=7.2e-10, Pearson correlation). To model interactions of stroma cell conditions and drug-induced apoptosis we established the following linear model: Viability ~ drug-effect + culture-model + drug-effect:culture-model. While activity of some drugs was significantly altered under co-culture conditions, we could also identify drugs with similar activity in mono- and co-cultures. For instance, the activity of common chemotherapeutics (fludarabine: p=0.002 at 0.6µM, cytarabine: p=0.001 at 1.5µM, ANOVA) or bromodomain inhibitors (I-BET-762: p=5.9e-5 at 4.5µM, JQ1: p=1.5e-8 at 1.5µM, ANOVA) was significantly reduced under co-culture conditions. In contrast, PI3K inhibitors idelalisib and duvelisib had a similar activity in mono-culture and stroma co-culture conditions and might represent a starting point to overcome stroma cell mediated drug resistance. In CLL, we identified IGHV mutation status and trisomy 12 as important determinants of response to kinase inhibitors. We confirmed these findings in stroma cell co-cultures, e.g. a better activity of B-cell receptor inhibitors in trisomy 12 and IGHV unmutated CLL. A systematic comparison of ex-vivo drug response pattern in mono- and co-cultures across 171 drug conditions will be presented. Conclusion: Our results suggest that high throughput co-culture drug testing can be robustly performed and provide an unprecedented understanding of how the stroma cell microenvironment and the genetic make-up of tumor cells contribute to drug resistance and sensitivity. Figure: Over 2 million microscopy images were acquired and analysed to assess drug resistance and sensitivity in a co-culture model of primary leukemia and bone marrow stroma cells. blue= Hoechst33342, green=Calcein AM, red=lysosomal dye NIR Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.