Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

Acute myeloid leukemia (AML) patients suffer dismal prognosis upon treatment resistance. To study functional heterogeneity of resistance, we generated serially transplantable patient-derived xenograft (PDX) models from one patient with AML and twelve clones thereof, each derived from a single stem cell, as proven by genetic barcoding. Transcriptome and exome sequencing segregated clones according to their origin from relapse one or two. Undetectable for sequencing, multiplex fluorochrome-guided competitive in vivo treatment trials identified a subset of relapse two clones as uniquely resistant to cytarabine treatment. Transcriptional and proteomic profiles obtained from resistant PDX clones and refractory AML patients defined a 16-gene score that was predictive of clinical outcome in a large independent patient cohort. Thus, we identified novel genes related to cytarabine resistance and provide proof of concept that intra-tumor heterogeneity reflects inter-tumor heterogeneity in AML.

Resistance against chemotherapy remains a major obstacle in treating patients with acute myeloid leukemia (AML). Novel therapeutic concepts are especially desired to target and eliminate resistant AML stem cells. Here we show that AML stem cells harbor the plasticity to switch from a low-cycling, chemotherapy resistant state into an actively proliferating state associated with treatment response. We used patient-derived xenograft (PDX) cells from patients with high risk or relapsed AML, which were lentivirally transduced for marker expression, stained with the proliferation-sensitive dye Carboxyfluorescein succinimidyl ester (CFSE), and re-transplanted into next-recipient mice. A rare subpopulation of AML cells displayed reduced proliferation in vivo , associated with increased treatment resistance. The proportion of AML cells with stem cell potential was identical in both, the highly and lowly proliferative sub-fraction. In re-transplantation experiments, proliferation behavior proved reversible, and AML stem cells were able to switch between a high and low proliferation state. Our data indicate that AML stem cells display functional plasticity in vivo , which might be exploited for therapeutic purposes, to prevent AML relapse and ultimately improve the prognosis of patients with AML.

Introduction: Acute myeloid leukemia (AML) shows substantial genetic and epigenetic heterogeneity, even within an individual patient. Due to treatment resistance and ability to induce relapse, adverse subclones present a major clinical challenge in determining the patient's prognosis. Here, we aimed at characterizing the genetic and functional heterogeneity within a single AML patient, and at identifying adverse subclones that result in therapy failure or give rise to relapse. Methods: Leukemic cells from an AML patient at first and second relapse were transplanted into immuno-compromised mice to generate patient-derived xenografts (PDX). PDX AML cells allowed serial transplantation and genetic engineering by lentiviruses. To distinguish single cells and generate PDX AML clones derived from a single cell (single cell clones, SCC), cells were transduced with a genetic barcode and transplanted into recipient mice near leukemia initiating cell frequency. Resulting SCC were genetically marked to express recombinant fluorochromes to enable flow cytometry analysis. All SCC were characterized for known subclonal mutations of the AML patient by targeted sequencing. Additionally, transcriptome and methylome analysis were performed by SCRB-seq and methylation array, respectively. Results: We successfully generated thirteen serially transplantable PDX SCC from a single AML patient, expressing combinations of up to four fluorochromes to enable competitive in vitro and in vivo experiments. In targeted sequencing, we found that SCC originated from at least four genetically distinct AML subclones and were distinguished by mutations in KRAS (4/13), NRAS (5/13), EZH2 (2/13) or EZH2 and NRAS (2/13). While the NRAS mutation was detected in a minority of bulk cells over serial passages (<10%) from both the first and second relapse, 50% of SCC carried the NRAS mutation. This indicates that NRAS mutated AML cells have an increased stem cell capacity upon transplantation of low cell numbers. Transcriptome analysis revealed 442 genes as differentially expressed between SCC and up to four biological replicates after adjustment for multiple hypothesis testing. Unsupervised clustering demonstrated a strong correlation of gene expression profiles with the respective genotype. In competitive in vivo experiments, homing capability was comparable between the four genetically distinct subclones. However, 2/2 EZH2-mutated SCC overgrew all other clones showing a clear growth advantage within two weeks of in vivo growth. The EZH2-mutated SCC (2/2) were resistant towards in vivo treatment with Cytarabine, whereas the KRAS (4/4), NRAS (5/5) mutated as well as the EZH2 and NRAS double-mutated SCC (2/2) responded to treatment. Conclusion: Taken together, we experimentally prove the existence of genetically and functionally diverse subclones within an individual AML sample. Our approach allows not only genetic, but also functional in vitro and in vivo characterization of adverse subclones. Our approach can be used to identify novel therapeutic approaches in order to specifically target the most adverse cells within patients' AML sample. Disclosures Metzeler: Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria.