Rewiring Cancer’s Compromised Cellular Circuitry

Cells contain intricate signaling circuits that tightly control all biological processes, including metabolism, growth, and survival. Cancers alter that wiring to emerge and grow, and in some cases, to resist treatment.

St. Jude scientists have been studying cellular signaling in depth to understand how malignant cells develop and evade death, revealing vulnerabilities in that compromised circuitry that can be targeted with novel therapeutic approaches.

Timing of cancer emergence in developing B cells impacts leukemia treatment

Even in cancers where most patients respond well to treatment, such as the most common childhood cancer, B-cell acute lymphoblastic leukemia (B-ALL), understanding how a patient’s specific form of the disease rewires cells has value. By uncovering these alterations, physicians can predict who will and will not respond to treatment and alter their approach.

Published in Nature Cancer, scientists at St. Jude and University Health Network’s Princess Margaret Cancer Centre in Toronto found that when a B cell becomes cancerous during development affects treatment outcomes, enriching researchers’ understanding of the cancer’s origin. They made this discovery by developing a robust single-cell reference atlas of normal human B-cell development and cross-referencing single-cell B-ALL data and outcomes data. They used that comparison to align the different B-ALL subtypes — and subgroups within subtypes — to different developmental stages, advancing fundamental understanding and association with outcomes. This enabled the researchers to find potentially actionable differences between types of B-ALL.

“At St. Jude, we have large acute lymphoblastic leukemia cohorts that have been genomically profiled with very rich underlying metadata, including outcomes,” said co-corresponding author Charles Mullighan, MBBS (Hons), MSc, MD, Comprehensive Cancer Center deputy director, Department of Pathology member.

Using that data, the researchers pinpointed the cell states in blood development from which B cells are diverted to become leukemia cells in patient samples. Many cancer samples arose from cells arrested in the pre-B and pro-B cell stages, which was anticipated, but there was much more diversity than expected. The data showed that less mature arrested cells retain features allowing lineage changes to occur, which can enable the cancer to evade therapy. They then developed those findings into a “multipotency score” that accurately predicted outcomes on a tested cohort of independent samples.

A vulnerability in an aggressive myeloproliferative neoplasm

While usually scientists look at how cancers emerge from normal development gone awry, they also study how existing cancer cells can be rewired to fuel a different and more aggressive form of the disease. This occurs in myeloproliferative neoplasms (MPNs), which develop when a person’s bone marrow creates too many of a specific blood cell type. These cancers have two phases, starting with a slow-moving chronic phase, then progressing to the acute blast phase (BP-MPN), with rapid growth of the neoplasm and a dismal prognosis, often just several months. The mechanisms of progression have been unclear, but massive genomic changes in chromosomes appear frequently in the blast phase.

St. Jude and University of Oxford scientists successfully untangled those massive genomic changes, finding that an amplification of a region of chromosome 21 was present in a subset of 64 patients with BP-MPN. Published in Nature Genetics, the researchers used CRISPR to individually study the genes within that region in BP-MPN cells, revealing that they depended on the gene DYRK1A.

“We uncovered that DYRK1A is driving chronic-MPNs into leukemia,” said corresponding author John Crispino, PhD, MBA, Division of Experimental Hematology director and Department of Hematology member. “By mapping how it rewired these cells, we also revealed a potential therapeutic vulnerability in BP-MPNs.”

The researchers found that DYRK1A indirectly upregulates BCL2, a gene that prevents cell death. When the researchers tested a BCL2 inhibitor, navitoclax, in combination with a DYRK1A inhibitor in cells, they saw substantial synergy, providing a new potential therapeutic approach.

We uncovered that DYRK1A is driving chronic-MPNs into leukemia. By mapping how it rewired these cells, we also revealed a potential therapeutic vulnerability in BP-MPNs.

John Crispino, PhD, MBA

Department of Hematology

A targetable mechanism behind a prevalent medulloblastoma predisposition gene

In contrast, researchers often already know that a mutant gene rewires cells to become malignant, but do not know how. This was the case for children who inherit a deficient ELP1 gene, which increases their risk of developing SHH-medulloblastoma, a subtype of malignant pediatric brain tumor. In Cancer Cell, St. Jude researchers uncovered how ELP1 deficiency leads cells to turn off the major cellular “circuit breaker” for cancer development, the tumor suppressor protein p53.

“We made the initial discovery of ELP1 deficiency predisposing children to SHH-medulloblastoma seven years ago, but now we’ve worked through its mechanism to an actual pharmacological intervention,” said co-corresponding author Paul Northcott, PhD, Center of Excellence in Neuro-Oncology Sciences director and Department of Developmental Neurobiology member. “We started with a gene in a spreadsheet and translated that into a potential targeted treatment.”

Protein p53 loss can fuel SHH-medulloblastoma. The researchers showed that ELP1 deficiency reduces p53 activity, putting neuronal progenitor cells at risk of developing into SHH-medulloblastoma. The protein MDM2 also reduces p53 activity by sparking the destruction of the p53 tumor suppressor. When the researchers prevented p53’s destruction by blocking MDM2, the increased p53 re-established its normal circuit breaker function, killing cancerous cells. MDM2 inhibition significantly increased the survival of mice implanted with ELP1-mutant SHH-medulloblastoma patient-derived xenografts, counteracting the effects of ELP1 loss.

“We showed pharmacologically restoring p53 activity using MDM2 inhibition is an exciting avenue toward a targeted therapy for ELP1-associated SHH-medulloblastoma,” Northcott said. The strategy is being evaluated for inclusion in clinical trials.

Mitochondria can drive cancer emergence and treatment resistance

Studying how cancers emerge can provide novel therapeutic opportunities. However, learning how cancers rewire themselves to resist treatments offers another compelling area of investigation to complement existing therapies and improve outcomes.

One unexpected source of treatment resistance comes from mitochondria, which power cells and have their own DNA (mtDNA) separate from the chromosomes and DNA in the nucleus. Until now, technical challenges have limited scientists’ understanding of how mtDNA mutations affect cancer growth.

“Each cell contains hundreds of copies of mitochondrial DNA. So, a mutation might be present at low levels in many cells, or at high levels in just a subset of cells,” explained corresponding author Mondira Kundu, MD, PhD, Department of Cell & Molecular Biology.

To overcome this challenge, Kundu’s team combined powerful computational tools, statistical analyses, bulk whole-genome sequencing, and single-cell studies. Published in Science Advances, the researchers first found that some mtDNA mutations occur before a B cell turns cancerous — and that these mutations are not always random. It appears that in some cases, cancer cells actively “select” for a mix of normal and mutated mitochondrial DNA.

Kundu’s team then deployed a computational tool, called NetBID2, created by Jiyang Yu, PhD, Department of Computational Biology interim chair. With this tool, the researchers discovered that certain mtDNA mutations are not only associated with activation of pathways linked to cell growth, which helps explain their selection, but also with pathways linked to resistance to glucocorticoids, a common therapy for ALL. Further analysis suggested that this type of mtDNA mutation may make leukemia cells more likely to resist treatment, opening a new area of investigation to leverage that information to help patients.

Each cell contains hundreds of copies of mitochondrial DNA. So, a mutation might be present at low levels in many cells, or at high levels in just a subset of cells.

Mondira Kundu, MD, PhD

Department of Cell & Molecular Biology

Overcoming menin treatment resistance in NUP98 leukemia subtypes

Instead of searching for cancer-related mutations, scientists sometimes already know the exact mutation that a malignancy uses to rewire cellular signaling; they just cannot target it due to the potential collateral damage to normal cells. This is true for many fusion oncoproteins, which comprise two abnormally joined proteins, thus giving the hybrid new functions that can promote cancer-driving genes. Because the two original proteins still exist in the body, targeting them directly can cause adverse effects. Scientists have looked for a workaround, targeting the proteins that fusions interact with instead.

Genomic rearrangements involving NUP98 result in a fusion protein often associated with poor prognosis and relapse in acute myeloid leukemia (AML). St. Jude and Dana-Farber Cancer Institute researchers systematically leveraged proteomic and CRISPR gene editing technologies to identify and study the proteins interacting with NUP98 fusions and DNA, which cannot be directly targeted, in AML models to find another vulnerability.

Published in Cancer Discovery, they identified the acetyltransferases MOZ/KAT6A and HBO1/KAT7, which help form a complex that activates pro-cancer gene expression. The scientists then combined MOZ/KAT6A and HBO1/KAT7 inhibitors with a menin inhibitor (menin works with NUP98 fusion proteins to foster leukemogenesis), greatly increasing survival in patient-derived mouse models.

“We found NUP98 fusions drive leukemia by assembling these proteins into a complex to switch on the expression of genes that turn normal cells into leukemia cells,” said corresponding author Mullighan. “We showed these inhibitors can stop the assembly of the switch, preventing activation of these cancer-driving genes, which is a novel therapeutic vulnerability in AML.”

Although most common in AML, NUP98 rearrangements have been linked to a wide spectrum of hematologic malignancies, including T-cell ALL and myelodysplastic neoplasms. How NUP98 fusion oncoproteins lead to such a broad range of diseases remains poorly understood, though it could have lessons for treatment.

Published in Blood, St. Jude researchers studied the genomic background of NUP98- rearranged leukemias. The team found that cooperating mutations, those which co-occur with specific NUP98 rearranged fusion oncoproteins, are linked to specific disease types. They then established models of NUP98-rearranged leukemias and showed that each NUP98 fusion oncoprotein induces blood cell lineages differentially, while cooperating alterations promote the survival of cells or block differentiation at distinct blood cell development stages.

“Our results show that we need to consider the impact of mutations beyond the driver fusion when studying these cancers,” said corresponding author Jeffery Klco, MD, PhD, Department of Pathology. “We need to think about potential resistance arising from sources outside of NUP98 rearrangements.”

Their models showed that cells with cooperating mutations may become less sensitive to menin inhibition. These findings indicate a need to view menin inhibitor effects through the lens of differentiation or cooperating mutations, to identify patients who are less likely to respond and guide alternative therapeutic strategies.

We showed these inhibitors can stop the assembly of the switch, preventing activation of these cancer-driving genes, which is a novel therapeutic vulnerability in AML.

Charles Mullighan, MBBS (Hons), MSc, MD

Department of Pathology

Finding the vulnerability in an AML treatment resistance pathway

Another set of difficult-to-treat AML cancers contains mutations in the genes IDH1 and IDH2. These mutations metabolically rewire cancer cells, resulting in a massive accumulation of a molecule known as an “oncometabolite” that helps fuel cancer growth, (R)-2-hydroxyglutarate (R-2HG).

AML often rapidly develops resistance to existing IDH inhibitors, resulting in a re-accumulation of R-2HG and further malignant growth. In Blood, St. Jude scientists reported how metabolic rewiring induced by mutant IDH was used to find a better target. They created cell lines representing the breadth of potential IDH mutations with CRISPR-gene editing, then profiled their gene expression to find a potential vulnerability.

“We consistently saw the cell surface protein CD44 upregulated in cells with IDH mutations,” said corresponding author Jian Xu, PhD, Department of Pathology. “That was exciting, because we could block CD44 and see its effect on this type of leukemia.”

When Xu’s group inhibited CD44 in leukemia cells with IDH mutations, they saw a significant decrease in cell growth, and a greater decrease when mixed with IDH inhibitors. They observed the same effect on xenografts of patient cancer samples, including those from patients who had developed resistance to IDH therapy in the clinic.

“Inhibiting CD44 and IDH at the same time can significantly enhance antileukemia activity, reducing leukemia burden and prolonging survival in these model systems,” Xu said. “This gives us confidence that the combination therapy could have the potential to outperform the single agent IDH inhibitor therapy currently used in the clinic, which suggests that it should be further evaluated.”

The right tools to study neuroblastoma’s wiring

Before testing new potential treatments, researchers need a model system that faithfully represents how the disease appears in people. For rare cancers, scientists create bespoke models to understand how they are driven by altered signaling in hopes of finding vulnerabilities. In the solid tumor neuroblastoma, approximately 50% of high-risk cases amplify the driver oncogene MYCN. However, “a subset of high-risk neuroblastoma patients do not have MYCN amplification in their cancer, but instead highly express the oncogene c-MYC,” said corresponding author Jun Yang, MD, PhD, Department of Surgery.

Yang found there was no research model available to study c-MYC-overexpressing neuroblastoma, so he created one, which he published in Cancer Research. To trigger the expression of c-MYC in neuroendocrine cells, the researchers used an enzyme called Cre recombinase, which is used to manipulate gene expression in specific cell types in model organisms. However, the model did not behave as expected.

“We observed tumor growth but found that it developed unexpectedly in the pancreas, which turned out to be a very rare neuroendocrine tumor called somatostatinoma,” Yang said. “For this model, we used tyrosine hydroxylase Cre, which is classically used to generate MYCN tumors. So, we tested other types of Cre lineages and, eventually, landed on an improved dopamine β-hydroxylase Cre. Finally, we got neuroblastoma tumor growth.”

The model successfully reflected key features of neuroblastoma, including sensitivity to drugs used to treat the disease. This gives researchers a better way to study this cancer and develop novel therapies.

Hijacking of embryonic development circuitry leads to retinoic acid sensitivity

Once a model is developed, most cancer research focuses on finding new vulnerabilities to target, but St. Jude scientists recently addressed the question in reverse for neuroblastoma. Decades ago, physicians found that adding retinoic acid to high-risk neuroblastoma treatment increased survival by 10%-15%, but how it was altering cancer cells’ circuitry to trigger their demise was unclear.

Intriguingly, the effect was only evident when retinoic acid was added to the later parts of therapy, after the main tumor had largely been eliminated. In a study published in Nature Communications, St. Jude scientists revealed why.

“We’ve come up with an explanation for a decades-long contradiction about why retinoic acid works in late, but not early, therapy,” said senior co-corresponding author Paul Geeleher, PhD, Department of Computational Biology. “Retinoic acid’s activity heavily depends on the cellular microenvironment.”

The researchers first found a subset of neuroblastoma cell lines that were extremely sensitive to retinoic acid. When they deleted candidate genes, the researchers found that bone morphogenetic proteins (BMPs), genes normally highly upregulated in the bone marrow that help drive embryonic bone and related tissue development, were responsible for that sensitivity. The scientists further uncovered how retinoic acid killed these cancer cells: If there are a lot of BMP-signaling pathway–related proteins already on the DNA, retinoic acid signaling combines with those proteins to “hijack” a normal development pathway and promote downstream cell death–related gene expression.

As the bone marrow microenvironment causes neuroblastoma cells to have higher BMP pathway activity, it neatly explains why retinoic acid is effective at treating metastasized cells in the bone marrow but not the primary tumors during up-front treatment.

Turning vulnerabilities in cancer’s wiring into tomorrow’s opportunities

These examples demonstrate that cancers have a variety of mechanisms to rewire cellular circuitry to promote their own growth and survival. However, St. Jude researchers are finding how those pathways are altered across pediatric malignancies, uncovering and testing novel therapeutic vulnerabilities introduced by that rewiring, moving one step closer to improving outcomes for children with these cancers.