Faculty Bibliography

UNLABELLED:Non-small lung cancers (NSCLC) frequently (∼30%) harbor KRAS driver mutations, half of which are KRASG12C. KRAS-mutant NSCLC with comutated STK11 and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapy. Development of KRASG12C inhibitors (G12Ci) provided a major therapeutic advance, but resistance still limits their efficacy. To identify genes whose deletion augments efficacy of the G12Cis adagrasib (MRTX-849) or adagrasib plus TNO155 (SHP2i), we performed genome-wide CRISPR/Cas9 screens on KRAS/STK11-mutant NSCLC lines. Recurrent, potentially targetable, synthetic lethal (SL) genes were identified, including serine-threonine kinases, tRNA-modifying and proteoglycan synthesis enzymes, and YAP/TAZ/TEAD pathway components. Several SL genes were confirmed by siRNA/shRNA experiments, and the YAP/TAZ/TEAD pathway was extensively validated in vitro and in mice. Mechanistic studies showed that G12Ci treatment induced gene expression of RHO paralogs and activators, increased RHOA activation, and evoked ROCK-dependent nuclear translocation of YAP. Mice and patients with acquired G12Ci- or G12Ci/SHP2i-resistant tumors showed strong overlap with SL pathways, arguing for the relevance of the screen results. These findings provide a landscape of potential targets for future combination strategies, some of which can be tested rapidly in the clinic. SIGNIFICANCE/UNASSIGNED:Identification of synthetic lethal genes with KRASG12C using genome-wide CRISPR/Cas9 screening and credentialing of the ability of TEAD inhibition to enhance KRASG12C efficacy provides a roadmap for combination strategies. See related commentary by Johnson and Haigis, p. 4005.

Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.

SHP2 (PTPN11) acts upstream of SOS1/2 to enable RAS activation. Allosteric SHP2 inhibitors (SHP2i) in the clinic prevent SHP2 activation, block proliferation of RTK- or cycling RAS mutant-driven cancers, and overcome "adaptive resistance." To identify SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive cell lines, recovering genes expected to cause resistance (NF1, PTEN, CDKN1B, LZTR1, and RASA2) and novel targets (INPPL1, MAP4K5, epigenetic modifiers). We screened 14 additional lines with a focused CRISPR library targeting common "hits" from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2/STK40 (6/14), and INPPL1 (5/14). INPPL1, MAP4K5, or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not lipid phosphatase activity. MAP4K5 acted upstream of MEK through a kinase-dependent target(s); LZTR1 had cell-dependent effects on RIT and RAS stability. INPPL1, MAP4K5, or LZTR1 deletion also conferred SHP2i resistance in vivo. Defining the SHP2i resistance landscape could suggest effective combination approaches.

UNLABELLED:. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)-key downstream effectors of LKB1- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE/CONCLUSIONS:tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant.

Intracellular oncoproteins can be inhibited with targeted therapy, but responses are not durable. Immune therapies can be curative, but most oncogene-driven tumors are unresponsive to these agents. Fragments of intracellular oncoproteins can act as neoantigens presented by the major histocompatibility complex (MHC) but recognizing minimal differences between oncoproteins and their normal counterparts is challenging. We have established a platform technology that exploits hapten-peptide conjugates generated by covalent inhibitors to create distinct neoantigens that selectively mark cancer cells. Using the FDA-approved covalent inhibitors sotorasib and osimertinib, we developed "HapImmuneTM" antibodies that bind to drug-peptide conjugate/MHC complexes but not to the free drugs. A HapImmuneTM-based bispecific T cell engager selectively and potently kills sotorasib-resistant lung cancer cells upon sotorasib treatment. Notably, it is effective against KRASG12C mutant cells with different HLA supertypes, HLA-A*02 and A*03/11, suggesting loosening of MHC restriction. Our strategy creates targetable neoantigens by design, unifying targeted and immune therapies.

Introduction: Myeloproliferative neoplasms (MPN) are myeloid malignancies with somatic JAK2, CALR or MPL mutations and constitutive activation of JAK2 signaling. JAK2 inhibitors show limited efficacy due to residual MAPK pathway activation. The molecular connection of JAK2 and MAPK pathway in MPN is not fully clarified. We study the role of SHP2, a protein tyrosine phosphatase involved in MAPK activation in other tyrosine kinase- driven malignancies, and assess its therapeutic potential.
Method(s): SHP2 was depleted by shRNA induced knockdown in Ba/F3 cells stably expressing Jak2V617F or wildtype Jak2. For SHP2 inhibition TNO155 and IACS13909 were used. Translational potential of JAK2/SHP2 inhibition was studied in Jak2V617F and MPLW515L mouse models.
Result(s): SHP2 was expressed at substantial levels in MPN cells including SET2, UKE-1 and Jak2V617F Ba/F3 cells. SHP2 knockdown reduced activation of MAPK pathway kinases including MEK, ERK and RSK as well as MAPK downstream effectors as DUSP6. SHP2 i nhibition w ith TNO155 o r I ACS-13909 analogously interfered with MAPK activation and effector expression and effects were most pronounced when JAK2 inhibition by ruxolitinib and SHP2 inhibition were combined (A). MPN cell proliferation was inhibited at significantly lower IC50 when combining ruxolitinib with TNO155 or IACS-13909 compared to ruxolitinib as single agent (B). Since inhibition of wildtype haematopoiesis is a concern, anti-proliferative activity of JAK2/SHP2 inhibition was assessed in cells with wildtype Jak2, which showed more modest inhibition (C). In a Jak2V617F mouse model, the SHP2 inhibitor TNO155 mediated corrective effects on MPN phenotype including splenomegaly, erythrocytosis and leucocytosis within a week. Of note, TNO155 as single agent showed similar effects as ruxolitinib at tolerable doses, while combined JAK2/SHP2 inhibition enhanced efficacy. In a MPLW515L mouse model with extensive leucocytosis, JAK2/SHP2 inhibitor treatment promptly normalized leukocyte counts which is not seen to this extent with ruxolitinib (D-E).
Conclusion(s): Our f indings suggest a s ignificant role of SHP2 function in MPN given enhanced MAPK suppression and corrective effects upon SHP2 targeting in MPN models. Further studies will delineate the involvement of phosphatase vs. nonphosphatase functions and address the potential of JAK2/SHP2 inhibition as therapeutic approach in MPN. (Figure Presented)

Despite being the most frequently altered oncogenic protein in solid tumours, KRAS has historically been considered 'undruggable' owing to a lack of pharmacologically targetable pockets within the mutant isoforms. However, improvements in drug design have culminated in the development of inhibitors that are selective for mutant KRAS in its active or inactive state. Some of these inhibitors have proven efficacy in patients with KRAS^G12C-mutant cancers and have become practice changing. The excitement associated with these advances has been tempered by drug resistance, which limits the depth and/or duration of responses to these agents. Improvements in our understanding of RAS signalling in cancer cells and in the tumour microenvironment suggest the potential for several novel combination therapies, which are now being explored in clinical trials. Herein, we provide an overview of the RAS pathway and review the development and current status of therapeutic strategies for targeting oncogenic RAS, as well as their potential to improve outcomes in patients with RAS-mutant malignancies. We then discuss challenges presented by resistance mechanisms and strategies by which they could potentially be overcome.

Single-nucleotide polymorphisms (SNPs) in RNF213, which encodes a 591-kD protein with AAA+ ATPase and RING E3 domains, are associated with a rare, autosomal dominant cerebrovascular disorder, moyamoya disease (MMD). MMD-associated SNPs primarily localize to the C-terminal region of RNF213, and some affect conserved residues in the RING domain. Although the autosomal dominant inheritance of MMD could most easily explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMD-associated SNPs on its E3 ligase activity have remained unclear. We found that RNF213 uses the E2-conjugating enzymes UBE2D2 and UBE2L3 to catalyze distinct ubiquitylation events. RNF213-UBED2 catalyzes K6 and, to a lesser extent, K48-dependent poly-ubiquitylation in vitro, whereas RNF213-UBE2L3 catalyzes K6-, K11-, and K48-dependent poly-ubiquitylation events. MMD-associated SNPs encode proteins with decreased E3 activity, and the most frequent MMD allele, RNF213

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTPs), a sub-population of cancer cells that survive via reversible, non-genetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKIs) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single cell RNA-sequencing reveal that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting show that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.