**7. Potential for new treatments by targeting copy number alterations**

SNVs have a proven track record of targetability using small molecules. Nonetheless, in the case of SOC, new cures are unlikely to be found unless somatic copy number alterations (SCNAs) are considered. Defining this interplay will be a difficult task. It remains unclear exactly which SCNAs are most critical to SOC proliferation and metastasis. The creation of cell line models will require new methods of whole-chromosome manipulation, even as attracting pharmaceutical company support will be harder due to limited experience which such targeting strategies, as well as conservative business approaches towards eventual clinical adaptation. Nonetheless, there are reasons to be optimistic that SCNA-targeted therapeutics can be effective and that some could enter the clinic in the near future.

Consider the abundance of SCNAs in advanced SOC relative to other cancer. The successful tumors have undergone selection. The phenotypes produced include well-known hallmarks of cancer: including cell cycle defects, heightened glucose uptake [106], spontaneous proliferative immortality [107], and dysregulated autophagy [108]. The same studies identify aneuploidyassociated characteristics which present vulnerabilities particular to these unstable cells. Perhaps the most promising vulnerability is an increased reliance on protein quality control processes such as ribosome biogenesis and maintenance factors and the cellular recycling process, autophagy. Aneuploid cells require these systems to function, and may result in a general reliance on catabolic function due to the proteotoxic effect of protein-complex subunit imbalance. Early studies recognized a general, if partial, sensitivity of these cells to rapamycin [106].

all pathway genes, they are typically less sensitive to such stresses, which opens therapeutic

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Given that proteostasis pathways are only one type of disruption caused by SCNAs in SOC, what other SCNA-disrupted pathways might be targetable? Alluring targets may include the proteins downstream of the E3 ligases commonly deleted in SOC; inhibition of cell-cycle regulators may selectively target SOC cells even without any mutation or than copy number changes. Strong amplification of peroxisome transporters and the glycerophospholipid metabolism pathways suggest that metabolic targeting may be worthwhile. *GSK3B* controls signaling between varying development and stem cells pathways [113], and was marked as the most cumulatively impactful amplified gene in SOC by HAPTRIG [8]. Interestingly, inhibition of GSK3β in preclinical models of SOC showed strong tumor inhibition [114], and a number of drugs targeting GSK3β are in development for cancer, diabetes, and neurodegeneration [115]. Finally, the 8q24 region is the most commonly amplified genomic region in SOC, containing MYC and FAK. Inhibitors to FAK, such as defactinib, are already being tested

in the clinic [116], and will hopefully provide some positive results in the near future.

disrupted pathways may be required to completely cure a patient.

A caveat of such designs is heterogeneity inherent in disease. Copy number instability is the result of SOC cells' extraordinary ability to create, tolerate, and expand genomic variation. Mathematical modeling of real tumor genetic data suggests that even small tumors with low mutation rates are statistically likely to contain multiple independent clones able to resist a particular drug treatment [117, 118]. Current SOC chemotherapeutics stimulate aneuploidy. Taxanes result in chromosome missegregation and platinum agents promote translocation events due to cross-strand DNA lesions. While it is absolutely true that common chemotherapeutics have limited combination potential due to dose limiting toxicities, that does not preclude the use of highly specific drugs to be used in combination or as maintenance therapy in SOC treatment regimens. Most likely, drugs independently targeting the many SCNA-

While most patients are caught late in the evolution of their disease, it may not be "too late" to treat them. A genomic analysis in highly metastatic recurrent SOC patients found that the tumors likely form a metastasis-to-metastasis spread [14]. This may explain why round after round of different chemotherapy can extend the life of SOC patients [119]. This implies that current chemotherapy is quite effective at destroying a great majority of cells, and the challenge that remains is how to complement it. The COAST strategy studied in our lab functioned equally well or better for cisplatin resistant forms of SOC [8]. Autophagy has been widely implicated in the ability of quiescent cells to survive, including in SOC [120], and has been directly shown to enable growth of Doxil resistant disease [121]. However, given that one COAST agent, chloroquine is often prescribed for the same patient for decades in highrisk malaria areas, while another, nelfinavir, is a daily long-term HIV medication with no serious side-effects, the use of COAST is warranted based on the decades of use of COAST drugs, in humans, for diseases other than cancer. The side effects are well established to be below current chemotherapeutics carboplatin, paclitaxel, and Doxil [111]. The greatest health concern may lie in kidney cells, which are also exquisitely sensitive to autophagy drugs [122]. Since the drugs target different but complementary pathways, it is feasible to design clinical trials involving either simultaneous treatment or sequential treatment, enabling a greater

windows for treatment.

Chromosome instability can incur resistance to taxanes, a common front line therapeutic for SOC [109]. Chromosomal instability endowed by docetaxel may in fact lead to subsequent additional chemoresistance [110], though it is clear that the resistant phenotype is at least initially offset by an increased sensitivity to carboplatin, the second primary chemotherapeutic co-administered with a taxane as standard of care. Although aneuploidy enables oncogenic characteristics, it offers targetable vulnerabilities as well.

The mapped SCNA patterns in SOC revealed a general fault in proteostasis control, centered on autophagy [8]. Yet these cells require autophagy to maintain viability. The delicate balance within SOC relative to normal tissue, appears to provide a therapeutic window for proteostasis-targeting agents. Since SOC cells are already severely disrupted in their proteostasis-regulatory mechanisms, further disturbance can greatly compromise survival even as normal cells readily process the insult. Given this premise, we developed the Combination of Autophagy Selective Therapeutics (COAST) method to effectively manage SOC in the lab [8]. The general approach involves directly stressing the proteostasis system, while inhibiting autophagy resolution (**Figure 4**).

Mice given the cocktail of five proteostasis drugs did not lose weight nor negatively alter their blood chemistry panel [111], tolerating these drugs for months of daily treatment [8]. In mouse models using recurrent human SOC cells, the proteostasis drugs out-performed platinum-taxane dual treatment. The results are consistent with previous approaches pursuing a "cyclops" hypothesis: that monoallelic deletions in cancer sensitize cancer cells to further disruption of that gene's function [112]. As normal cells bear a full complement of

**Figure 4.** SCNA can compromise key cellular infrastructure. The figure shows how the disruption of autophagy capacity that is observed in SOC can render the cells more sensitive to agents that modulate autophagy. The lack of genetic infrastructure, combined with a constant requirement for autophagy, opens a therapeutic window for these agents. Nonetheless, the heterogeneity within tumors and between patients insures that no single agent, used alone, will provide a complete benefit.

all pathway genes, they are typically less sensitive to such stresses, which opens therapeutic windows for treatment.

Given that proteostasis pathways are only one type of disruption caused by SCNAs in SOC, what other SCNA-disrupted pathways might be targetable? Alluring targets may include the proteins downstream of the E3 ligases commonly deleted in SOC; inhibition of cell-cycle regulators may selectively target SOC cells even without any mutation or than copy number changes. Strong amplification of peroxisome transporters and the glycerophospholipid metabolism pathways suggest that metabolic targeting may be worthwhile. *GSK3B* controls signaling between varying development and stem cells pathways [113], and was marked as the most cumulatively impactful amplified gene in SOC by HAPTRIG [8]. Interestingly, inhibition of GSK3β in preclinical models of SOC showed strong tumor inhibition [114], and a number of drugs targeting GSK3β are in development for cancer, diabetes, and neurodegeneration [115]. Finally, the 8q24 region is the most commonly amplified genomic region in SOC, containing MYC and FAK. Inhibitors to FAK, such as defactinib, are already being tested in the clinic [116], and will hopefully provide some positive results in the near future.

A caveat of such designs is heterogeneity inherent in disease. Copy number instability is the result of SOC cells' extraordinary ability to create, tolerate, and expand genomic variation. Mathematical modeling of real tumor genetic data suggests that even small tumors with low mutation rates are statistically likely to contain multiple independent clones able to resist a particular drug treatment [117, 118]. Current SOC chemotherapeutics stimulate aneuploidy. Taxanes result in chromosome missegregation and platinum agents promote translocation events due to cross-strand DNA lesions. While it is absolutely true that common chemotherapeutics have limited combination potential due to dose limiting toxicities, that does not preclude the use of highly specific drugs to be used in combination or as maintenance therapy in SOC treatment regimens. Most likely, drugs independently targeting the many SCNAdisrupted pathways may be required to completely cure a patient.

While most patients are caught late in the evolution of their disease, it may not be "too late" to treat them. A genomic analysis in highly metastatic recurrent SOC patients found that the tumors likely form a metastasis-to-metastasis spread [14]. This may explain why round after round of different chemotherapy can extend the life of SOC patients [119]. This implies that current chemotherapy is quite effective at destroying a great majority of cells, and the challenge that remains is how to complement it. The COAST strategy studied in our lab functioned equally well or better for cisplatin resistant forms of SOC [8]. Autophagy has been widely implicated in the ability of quiescent cells to survive, including in SOC [120], and has been directly shown to enable growth of Doxil resistant disease [121]. However, given that one COAST agent, chloroquine is often prescribed for the same patient for decades in highrisk malaria areas, while another, nelfinavir, is a daily long-term HIV medication with no serious side-effects, the use of COAST is warranted based on the decades of use of COAST drugs, in humans, for diseases other than cancer. The side effects are well established to be below current chemotherapeutics carboplatin, paclitaxel, and Doxil [111]. The greatest health concern may lie in kidney cells, which are also exquisitely sensitive to autophagy drugs [122].

Since the drugs target different but complementary pathways, it is feasible to design clinical trials involving either simultaneous treatment or sequential treatment, enabling a greater

**Figure 4.** SCNA can compromise key cellular infrastructure. The figure shows how the disruption of autophagy capacity that is observed in SOC can render the cells more sensitive to agents that modulate autophagy. The lack of genetic infrastructure, combined with a constant requirement for autophagy, opens a therapeutic window for these agents. Nonetheless, the heterogeneity within tumors and between patients insures that no single agent, used alone, will

reliance on catabolic function due to the proteotoxic effect of protein-complex subunit imbalance. Early studies recognized a general, if partial, sensitivity of these cells to rapamycin [106]. Chromosome instability can incur resistance to taxanes, a common front line therapeutic for SOC [109]. Chromosomal instability endowed by docetaxel may in fact lead to subsequent additional chemoresistance [110], though it is clear that the resistant phenotype is at least initially offset by an increased sensitivity to carboplatin, the second primary chemotherapeutic co-administered with a taxane as standard of care. Although aneuploidy enables oncogenic

The mapped SCNA patterns in SOC revealed a general fault in proteostasis control, centered on autophagy [8]. Yet these cells require autophagy to maintain viability. The delicate balance within SOC relative to normal tissue, appears to provide a therapeutic window for proteostasis-targeting agents. Since SOC cells are already severely disrupted in their proteostasis-regulatory mechanisms, further disturbance can greatly compromise survival even as normal cells readily process the insult. Given this premise, we developed the Combination of Autophagy Selective Therapeutics (COAST) method to effectively manage SOC in the lab [8]. The general approach involves directly stressing the proteostasis system,

Mice given the cocktail of five proteostasis drugs did not lose weight nor negatively alter their blood chemistry panel [111], tolerating these drugs for months of daily treatment [8]. In mouse models using recurrent human SOC cells, the proteostasis drugs out-performed platinum-taxane dual treatment. The results are consistent with previous approaches pursuing a "cyclops" hypothesis: that monoallelic deletions in cancer sensitize cancer cells to further disruption of that gene's function [112]. As normal cells bear a full complement of

characteristics, it offers targetable vulnerabilities as well.

124 Ovarian Cancer - From Pathogenesis to Treatment

while inhibiting autophagy resolution (**Figure 4**).

provide a complete benefit.

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