Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues ...that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.
Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has ...been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader. Graphical overview.
The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less ...well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34
hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.
Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal ...feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in
. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.
In this work, crystal structures of commercially available photochromic compounds, i.e., spiropyrans and spirooxazines, were investigated by single-crystal X-ray diffraction. A total of five new ...structures were obtained via isothermal evaporation experiments under different conditions, namely 1,3,3-Trimethylindolino-benzopyrylospiran (I), 1,3,3-Trimethylindolinonaphtospirooxaxine (II), 1-(2-Hydroxyethyl)-3,3-dimethylindolino-6′-nitrobenzopyrylospiran (III), and 1,3,3-Trimethylindolino-8′-methoxybenzopyrylospiran (IVa and IVb). Since the basic structure of a spiropyran/-oxazine does not present typical hydrogen bond accepting and donating groups, this study illustrates the importance of additional functional groups connected to this kind of molecules to induce specific intermolecular interactions. Our results show that possible hydrogen bonding interactions are rather weak due to the high steric demand of these compounds. These results are supported by a search of the Cambridge structural database focused on related structures.
Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were ...significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34
hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.
Abstract only
Introduction
Tight control of metabolic processes is crucial for normal hematopoietic cell development, proliferation and cancer cell expansion. These processes all require variable ...bioenergetic demands, which are in part met by an upregulation of glycolytic activity. Hexokinases (HK1, 2, 3 and 4) catalyze the first and irreversible step of glycolysis. Recent evidence implicates a tumor promoting role for HK2 in KRas‐driven lung and ErbB2‐driven breast cancer. In contrast to the ubiquitously expressed HK1 and 2 in mammalian cells, HK3 is predominantly expressed in hematopoietic cells. We have identified HK3 as a transcriptional target of the hematopoietic‐specific transcription factor, PU.1 (SPI‐1). Moreover, HK3 is downregulated in acute myeloid leukemia (AML) patient samples and upregulated during terminal differentiation of selected acute myeloid leukemia cell lines concomitant with PU.1 upregulation.
Methods and Results
In primary human CD34
+
hematopoietic stem and early progenitor cells (HSPCs), we found HK1‐3 to be present at similar levels. While HK1 and HK2 mRNA levels remained unchanged during HSPC myeloid differentiation, HK3 mRNA levels significantly increased. Given the oncogenic role of HK2 and our findings of HK3 in AML cell lines, we next evaluated whether unique roles exist for HK2 and 3 in myeloid cells. Using shRNA or CRISPR/CAS9 technology, we generated HK2 and HK3 knockdowns (KD) and knockouts (KO) in HL60 and NB4 AML cell lines. Assessment of glycolytic and oxidative metabolism in HK altered cells revealed that loss of HK3 had no effect on steady state glycolytic activity, while loss of HK2 drastically reduced steady state glycolysis. In contrast, loss of HK3 expression resulted in increased cell death during all‐trans retinoic acid (ATRA) induced differentiation of HL60 (86±2% vs 36±3% viable cells at day 4, p<0.0001). Furthermore, investigation of HK3 subcellular localization point towards both cytosolic and nuclear localization for ectopically expressed HK3 in HL60 cells by western blot analysis. Mass spectrometric (MS) analysis of HL60 HK2 KO lines identified upregulation of proteins involved in key signaling pathways (Wnt, EGF and PDGF), as well as macromolecular metabolic processes, Fig.
1A
. In contrast, HK3 KO HL60 cells showed upregulation of proteins involved in cellular homeostatic processes (ubiquitin proteasome pathway) and cancer‐related signaling pathways (Wnt, Ras) (p‐value<0.05), Fig.
1B
.
Discussion
Together our findings implicate HK2 as the major glycolytically active isoform of hexokinases in myeloid cell lines, whereas HK3 exerts non‐canonical regulatory functions impacting normal myeloid differentiation and cancer regulation. Investigations are underway characterizing the metabolic phenotype of HK KO AML cells as well as defining the precise roles of HK2 and HK3 in normal HSPC differentiation, proliferation and metabolic activity.
Support or Funding Information
Swiss Cancer Research
Panther Pathway analyses of significantly upregulated proteins identified by MS analyses upon loss of either HK2 (
A
) or HK3 (
B
) in HL60 cell lines evaluated at steady state.
Figure 1
Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an ...adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein.
We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively.
To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity.
Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation.
No relevant conflicts of interest to declare.
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