UPR, autophagy, and mitochondria crosstalk underlies the ER stress response.

Cellular stress, induced by external or internal cues, activates several well-orchestrated processes aimed at either restoring cellular homeostasis or committing to cell death. Those processes include the unfolded protein response (UPR), autophagy, hypoxia, and mitochondrial function, which are part of the global endoplasmic reticulum (ER) stress (ERS) response. When one of the ERS elements is impaired, as often occurs under pathological conditions, overall cellular homeostasis may be perturbed. Further, activation of the UPR could trigger changes in mitochondrial function or autophagy, which could modulate the UPR, exemplifying crosstalk processes. Among the numerous factors that control the magnitude or duration of these processes are ubiquitin ligases, which govern overall cellular stress outcomes. Here we summarize crosstalk among the fundamental processes governing ERS responses.

X-linked inhibitor of apoptosis protein represents a promising therapeutic target for relapsed/refractory ALL.

Acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children, and relapse/refractory (r/r) disease is difficult to treat, both in children and adults. In search for novel treatment options against r/r ALL, we studied inhibitor of apoptosis proteins (IAP) and Smac mimetics (SM). SM-sensitized r/r ALL cells towards conventional chemotherapy, even upon resistance against SM alone. The combination of SM and chemotherapy-induced cell death via caspases and PARP, but independent from cIAP-1/2, RIPK1, TNFα or NF-κB. Instead, XIAP was identified to mediate SM effects. Molecular manipulation of XIAP in vivo using microRNA-30 flanked shRNA expression in cell lines and patient-derived xenograft (PDX) models of r/r ALL mimicked SM effects and intermediate XIAP knockdown-sensitized r/r ALL cells towards chemotherapy-induced apoptosis. Interestingly, upon strong XIAP knockdown, PDX r/r ALL cells were outcompeted in vivo, even in the absence of chemotherapy. Our results indicate a yet unknown essential function of XIAP in r/r ALL and reveal XIAP as a promising therapeutic target for r/r ALL.

In vivo PDX CRISPR/Cas9 screens reveal mutual therapeutic targets to overcome heterogeneous acquired chemo-resistance.

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.

A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models.

CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.

Tumor Cell Dormancy-Triggered by the Niche.

Dormant cancer cells often survive treatment and increase the risk for tumor relapse, associated with dismal prognosis. Two recent papers describe mechanisms used by the bone marrow niche to regulate leukemia dormancy. The findings provide a molecular basis for niche-targeting therapies that may enable elimination of dormant tumor cells.

A rare subgroup of leukemia stem cells harbors relapse-inducing potential in acute lymphoblastic leukemia.

After initially successful chemotherapy, relapse frequently jeopardizes the outcome of patients with acute leukemia. Because of their adverse characteristics of self-renewal and dormancy, leukemia stem cells have been hypothesized to play a critical role in resistance to antiproliferative chemotherapy and the development of relapse. The high abundance of stem-like cells in acute lymphoblastic leukemia (ALL), however, suggests that not all leukemia-initiating cells carry these adverse characteristics, complicating the biological characterization of relapse-inducing cells in this malignancy. Here, we review sources of therapy resistance and relapse in acute leukemias, which include tumor cell plasticity and reversible characteristics. We discuss the development of patient-derived mouse models that are genetically engineered to mimic long-term dormancy and minimal residual disease in patients. These models allow the tracking and functional characterization of patient-derived ALL blasts that combine the properties of long-term dormancy, treatment resistance, and stemness. Finally, we discuss possible therapeutic avenues to target the functional plasticity of leukemia-initiating cells in ALL.

Ubiquitin Ligases in Cancer Immunotherapy - Balancing Antitumor and Autoimmunity.

Considerable progress has been made in understanding the contribution of E3 ubiquitin ligases to health and disease, including the pathogenesis of immunological disorders. Ubiquitin ligases exert exquisite spatial and temporal control over protein stability and function, and are thus crucial for the regulation of both innate and adaptive immunity. Given that immune responses can be both detrimental (autoimmunity) and beneficial (antitumor immunity), it is vital to understand how ubiquitin ligases maintain immunological homeostasis. Such knowledge could reveal novel mechanisms underlying immune regulation and identify new therapeutic approaches to enhance antitumor immunity and safeguard against autoimmunity.