Hooman Hefzi
@hefzi-lab.bsky.social
PI of the Advanced Mammalian Cell Engineering Lab at the Technical University of Denmark
http://bioengineering.dtu.dk/amce
http://bioengineering.dtu.dk/amce
High on the list of things I'd love to explore!
February 10, 2025 at 8:23 PM
High on the list of things I'd love to explore!
I am also really glad that people far more creative than I dressed up a hamster as Godzilla so that when I decided to name the cell line CHO ZeLa (for Zero Lacate) I could use lots of fun pictures for presentations.
January 20, 2025 at 9:41 AM
I am also really glad that people far more creative than I dressed up a hamster as Godzilla so that when I decided to name the cell line CHO ZeLa (for Zero Lacate) I could use lots of fun pictures for presentations.
Finally, this work could not have been done without the tireless work of 30+ other authors from 7+ institutions over ~8 years! Special shout outs to @natelewis3.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
January 20, 2025 at 9:41 AM
Finally, this work could not have been done without the tireless work of 30+ other authors from 7+ institutions over ~8 years! Special shout outs to @natelewis3.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
I've recently started as an Associate Professor at the Technical University of Denmark and my new research group is definitely interested in poking at this in more detail :) It's a big space so reach out if you think a collaboration could be fun!
January 20, 2025 at 9:41 AM
I've recently started as an Associate Professor at the Technical University of Denmark and my new research group is definitely interested in poking at this in more detail :) It's a big space so reach out if you think a collaboration could be fun!
Sosososo many interesting future lines of inquiry. I can't say we've figured out the "why" of the Warburg effect, but I think it is safe to say that it is definitely dispensable for proliferation.
January 20, 2025 at 9:41 AM
Sosososo many interesting future lines of inquiry. I can't say we've figured out the "why" of the Warburg effect, but I think it is safe to say that it is definitely dispensable for proliferation.
Strikingly, compared to a human Warburg-null LS174T cancer line (LDHA/B double KO with reduced growth), we saw almost no overlap in consistently, differentially expressed genes (vs WT); in fact, more genes showed consistent opposite directionality in CHO Warburg-null cells.
January 20, 2025 at 9:41 AM
Strikingly, compared to a human Warburg-null LS174T cancer line (LDHA/B double KO with reduced growth), we saw almost no overlap in consistently, differentially expressed genes (vs WT); in fact, more genes showed consistent opposite directionality in CHO Warburg-null cells.
Metabolic genes linked to redox metabolism, however, did seem generally perturbed.
January 20, 2025 at 9:41 AM
Metabolic genes linked to redox metabolism, however, did seem generally perturbed.
When we looked at RNA-Seq to identify an underlying basis for the Warburg-null phenotype, we were surprised to see very little overlap in differentially expressed genes (vs WT) when looking at Warburg-null clones derived from 2 different CHO cell lines.
January 20, 2025 at 9:41 AM
When we looked at RNA-Seq to identify an underlying basis for the Warburg-null phenotype, we were surprised to see very little overlap in differentially expressed genes (vs WT) when looking at Warburg-null clones derived from 2 different CHO cell lines.
Unsurprisingly, Warburg-null cells:
• Used slightly more oxygen
• Showed increased glucose-->TCA cycle flux
• Were more sensitive to mitochondrial inhibitors
• Showed a significantly lower NAD/NADH ratio
• Used slightly more oxygen
• Showed increased glucose-->TCA cycle flux
• Were more sensitive to mitochondrial inhibitors
• Showed a significantly lower NAD/NADH ratio
January 20, 2025 at 9:41 AM
Unsurprisingly, Warburg-null cells:
• Used slightly more oxygen
• Showed increased glucose-->TCA cycle flux
• Were more sensitive to mitochondrial inhibitors
• Showed a significantly lower NAD/NADH ratio
• Used slightly more oxygen
• Showed increased glucose-->TCA cycle flux
• Were more sensitive to mitochondrial inhibitors
• Showed a significantly lower NAD/NADH ratio
We confirmed that the Warburg-null CHO cells could still be used to make biotherapeutic proteins in high quantity and quality and turned to investigate how Warburg-null cells responded to elimination of this ubiquitous phenotype.
January 20, 2025 at 9:41 AM
We confirmed that the Warburg-null CHO cells could still be used to make biotherapeutic proteins in high quantity and quality and turned to investigate how Warburg-null cells responded to elimination of this ubiquitous phenotype.
We later showed that this same approach was effective in another CHO cell line as well as HEK293, making us think that this may be a generalizable genetic engineering strategy to eliminate the Warburg effect!
January 20, 2025 at 9:41 AM
We later showed that this same approach was effective in another CHO cell line as well as HEK293, making us think that this may be a generalizable genetic engineering strategy to eliminate the Warburg effect!
We later made a range of knockouts and showed that knocking out even 1 (of 4) pyruvate dehydrogenase kinases enabled the knockout of Ldha and elimination of the Warburg effect. These cells grew as well as wildtype, did not produce lactate, and consumed much less glucose.
January 20, 2025 at 9:41 AM
We later made a range of knockouts and showed that knocking out even 1 (of 4) pyruvate dehydrogenase kinases enabled the knockout of Ldha and elimination of the Warburg effect. These cells grew as well as wildtype, did not produce lactate, and consumed much less glucose.
We first generated knockouts of all pyruvate dehydrogenase kinases and Ldha. We confirmed that lactate dehydrogenase is not detected, that cells do not produce lactate, and show negligible lactate dehydrogenase activity (wildtype in blue, knockout lines in red).
January 20, 2025 at 9:41 AM
We first generated knockouts of all pyruvate dehydrogenase kinases and Ldha. We confirmed that lactate dehydrogenase is not detected, that cells do not produce lactate, and show negligible lactate dehydrogenase activity (wildtype in blue, knockout lines in red).
~8(!) years ago, we tried to eliminate the Warburg effect in CHO cells and found that simultaneous knock out of pyruvate dehydrogenase kinases and lactate dehydrogenases eliminates lactate production w/out impacting growth!
January 20, 2025 at 9:41 AM
~8(!) years ago, we tried to eliminate the Warburg effect in CHO cells and found that simultaneous knock out of pyruvate dehydrogenase kinases and lactate dehydrogenases eliminates lactate production w/out impacting growth!
Thus, significant effort has been dedicated to eliminate lactate production in these lines, but none have proven effective. This is consistent with observations that targeting lactate production in cancer leads to cell death, though this has not led to approved therapeutics.
January 20, 2025 at 9:41 AM
Thus, significant effort has been dedicated to eliminate lactate production in these lines, but none have proven effective. This is consistent with observations that targeting lactate production in cancer leads to cell death, though this has not led to approved therapeutics.
Mammalian lines such as Chinese hamster ovary (CHO) cells and HEK293 are broadly used for the production of life-saving biotherapeutics, and also exhibit the Warburg effect. Lactate accumulation in cell culture is detrimental to growth and, thus, biotherapeutic production.
January 20, 2025 at 9:41 AM
Mammalian lines such as Chinese hamster ovary (CHO) cells and HEK293 are broadly used for the production of life-saving biotherapeutics, and also exhibit the Warburg effect. Lactate accumulation in cell culture is detrimental to growth and, thus, biotherapeutic production.
Over the years, there has been an incredible amount of research trying to answer the "why" of this phenomenon. Some theorized that it was necessary for rapid ATP generation or to provide the necessary precursors for biosynthetic pathways.
January 20, 2025 at 9:41 AM
Over the years, there has been an incredible amount of research trying to answer the "why" of this phenomenon. Some theorized that it was necessary for rapid ATP generation or to provide the necessary precursors for biosynthetic pathways.
101 years ago, Otto Warburg observed that cancer cells produced high quantities of lactate from glucose, even in the presence of oxygen. Later research suggested that this "Warburg effect" was a hallmark of all proliferative mammalian cells, e.g., stem cells.
January 20, 2025 at 9:41 AM
101 years ago, Otto Warburg observed that cancer cells produced high quantities of lactate from glucose, even in the presence of oxygen. Later research suggested that this "Warburg effect" was a hallmark of all proliferative mammalian cells, e.g., stem cells.
I am also really glad that people far more creative than I dressed up a hamster as Godzilla so that when I decided to name the cell line CHO ZeLa (for Zero Lacate) I could use lots of fun pictures for presentations.
January 20, 2025 at 9:32 AM
I am also really glad that people far more creative than I dressed up a hamster as Godzilla so that when I decided to name the cell line CHO ZeLa (for Zero Lacate) I could use lots of fun pictures for presentations.
Finally, this work could not have been done without the tireless work of 30+ other authors from 7+ institutions over ~8 years! Special shout outs to
@nate3791.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
@nate3791.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
January 20, 2025 at 9:32 AM
Finally, this work could not have been done without the tireless work of 30+ other authors from 7+ institutions over ~8 years! Special shout outs to
@nate3791.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
@nate3791.bsky.social and Bjørn Voldborg for humoring me when I asked them to knock out these 5 genes at once just to give it a shot.
I've recently started as an Associate Professor at the Technical University in Denmark and my new research group is definitely interested in poking at this in more detail :) It's a big space so reach out if you think a collaboration could be fun!
January 20, 2025 at 9:32 AM
I've recently started as an Associate Professor at the Technical University in Denmark and my new research group is definitely interested in poking at this in more detail :) It's a big space so reach out if you think a collaboration could be fun!
Sosososo many interesting future lines of inquiry. I can't say we've figured out the "why" of the Warburg effect, but I think it is safe to say that it is definitely dispensable for proliferation.
January 20, 2025 at 9:32 AM
Sosososo many interesting future lines of inquiry. I can't say we've figured out the "why" of the Warburg effect, but I think it is safe to say that it is definitely dispensable for proliferation.
Strikingly, compared to a human Warburg-null LS174T cancer line (LDHA/B double KO with reduced growth), we saw almost no overlap in consistently, differentially expressed genes (vs WT); in fact, more genes showed consistent opposite directionality in CHO Warburg-null cells.
January 20, 2025 at 9:32 AM
Strikingly, compared to a human Warburg-null LS174T cancer line (LDHA/B double KO with reduced growth), we saw almost no overlap in consistently, differentially expressed genes (vs WT); in fact, more genes showed consistent opposite directionality in CHO Warburg-null cells.